Compositions and methods of treating muscle atrophy and myotonic dystrophy

ABSTRACT

Disclosed herein are polynucleic acid molecules, pharmaceutical compositions, and methods for treating muscle atrophy or myotonic dystrophy.

CROSS-REFERENCE

This application is a divisional of U.S. application Ser. No.17/024,624, filed on Sep. 17, 2020, which is a continuation of U.S.application Ser. No. 16/435,422, filed Jun. 7, 2019, which is acontinuation of PCT Application No. PCT/US2018/064359, filed Dec. 6,2018, which claims priority to U.S. Provisional Application No.62/595,545, filed Dec. 6, 2017, and U.S. Provisional Application No.62/725,883, filed Aug. 31, 2018, which each of the applications isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 7, 2019, isnamed 45532-722_405_SL.txt and is 3,143,531 bytes in size.

BACKGROUND OF THE DISCLOSURE

Gene suppression by RNA-induced gene silencing provides several levelsof control: transcription inactivation, small interfering RNA(siRNA)-induced mRNA degradation, and siRNA-induced transcriptionalattenuation. In some instances, RNA interference (RNAi) provides longlasting effect over multiple cell divisions. As such, RNAi represents aviable method useful for drug target validation, gene function analysis,pathway analysis, and disease therapeutics.

SUMMARY OF THE DISCLOSURE

Disclosed herein, in certain embodiments, are polynucleic acid moleculesand pharmaceutical compositions for modulating a gene associated withmuscle atrophy (or an atrogene). In some embodiments, also describedherein are methods of treating muscle atrophy with a polynucleic acidmolecule or a polynucleic acid molecule conjugate disclosed herein.

Disclosed herein, in certain embodiments, is a molecule of Formula (I):A-X₁—B—X₂—C (Formula I) wherein, A is a binding moiety; B is apolynucleotide that hybridizes to a target sequence of an atrogene; C isa polymer; and X₁ and X₂ are each independently selected from a bond ora non-polymeric linker; wherein the polynucleotide comprises at leastone 2′ modified nucleotide, at least one modified internucleotidelinkage, or at least one inverted abasic moiety; and wherein A and C arenot attached to B at the same terminus. In some embodiments, theatrogene comprises a differentially regulated (e.g., an upregulated ordownregulated) gene within the IGF1-Akt-FoxO pathway, theglucocorticoids-GR pathway, the PGC1α-FoxO pathway, the TNFα-NFκBpathway, or the myostatin-ActRIIb-Smad2/3 pathway. In some embodiments,the atrogene encodes an E3 ligase. In some embodiments, the atrogeneencodes a Forkhead box transcription factor. In some embodiments, theatrogene comprises atrogin-1 gene (FBXO32), MuRF1 gene (TRIM63), FOXO1,FOXO3, or MSTN. In some embodiments, the atrogene comprises DMPK. Insome embodiments, B consists of a polynucleotide that hybridizes to atarget sequence of an atrogene. In some embodiments, C consists of apolymer. In some embodiments, the at least one 2′ modified nucleotidecomprises 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl,2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA) modified nucleotide. In someembodiments, the at least one 2′ modified nucleotide comprises lockednucleic acid (LNA) or ethylene nucleic acid (ENA). In some embodiments,the at least one modified internucleotide linkage comprises aphosphorothioate linkage or a phosphorodithioate linkage. In someembodiments, the at least one inverted abasic moiety is at at least oneterminus. In some embodiments, the polynucleotide comprises a singlestrand which hybridizes to the target sequence of an atrogene. In someembodiments, the polynucleotide comprises a first polynucleotide and asecond polynucleotide hybridized to the first polynucleotide to form adouble-stranded polynucleic acid molecule, wherein either the firstpolynucleotide or the second polynucleotide also hybridizes to thetarget sequence of an atrogene. In some embodiments, the secondpolynucleotide comprises at least one modification. In some embodiments,the first polynucleotide and the second polynucleotide are RNAmolecules. In some embodiments, the polynucleotide hybridizes to atleast 8 contiguous bases of the target sequence of an atrogene. In someembodiments, the polynucleotide comprises a sequence that is at least60%, 70%, 80%, 85%, 90%, 95%, or 99% complementary to a sequence as setforth in SEQ ID NOs: 28-141, 370-480, and 703-3406. In some embodiments,the polynucleotide is between about 8 and about 50 nucleotides inlength. In some embodiments, the polynucleotide is between about 10 andabout 30 nucleotides in length. In some embodiments, the firstpolynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth inSEQ ID NOs: 142-255, 256-369, 481-591, 592-702, and 3407-14222. In someembodiments, the second polynucleotide comprises a sequence having atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto a sequence as set forth in SEQ ID NOs: 142-255, 256-369, 481-591,592-702, and 3407-14222. In some embodiments, X₁ and X₂ areindependently a C₁-C₆ alkyl group. In some embodiments, X₁ and X₂ areindependently a homobifuctional linker or a heterobifunctional linker,optionally conjugated to a C₁-C₆ alkyl group. In some embodiments, A isan antibody or binding fragment thereof. In some embodiments, Acomprises a humanized antibody or binding fragment thereof, chimericantibody or binding fragment thereof, monoclonal antibody or bindingfragment thereof, monovalent Fab′, divalent Fab2, single-chain variablefragment (scFv), diabody, minibody, nanobody, single-domain antibody(sdAb), or camelid antibody or binding fragment thereof. In someembodiments, A is an anti-transferrin receptor antibody or bindingfragment thereof. In some embodiments, C is polyethylene glycol. In someembodiments, A-X₁ is conjugated to the 5′ end of B and X₂—C isconjugated to the 3′ end of B. In some embodiments, X₂—C is conjugatedto the 5′ end of B and A-X₁ is conjugated to the 3′ end of B. In someembodiments, A is directly conjugated to X₁. In some embodiments, C isdirectly conjugated to X₂. In some embodiments, B is directly conjugatedto X₁ and X₂. In some embodiments, the molecule further comprises D. Insome embodiments, D is conjugated to C or to A. In some embodiments, Dis an endosomolytic polymer.

Disclosed herein, in certain embodiments, is a polynucleic acid moleculeconjugate comprising a binding moiety conjugated to a polynucleotidethat hybridizes to a target sequence of an atrogene; wherein thepolynucleotide optionally comprises at least one 2′ modified nucleotide,at least one modified internucleotide linkage, or at least one invertedabasic moiety; and wherein the polynucleic acid molecule conjugatemediates RNA interference against the atrogene, thereby treating muscleatrophy in a subject. In some embodiments, the atrogene comprises adifferentially regulated (e.g., an upregulated or downregulated) genewithin the IGF1-Akt-FoxO pathway, the glucocorticoids-GR pathway, thePGC1α-FoxO pathway, the TNFα-NFκB pathway, or themyostatin-ActRIIb-Smad2/3 pathway. In some embodiments, the atrogeneencodes an E3 ligase. In some embodiments, the atrogene encodes aForkhead box transcription factor. In some embodiments, the atrogenecomprises ligand of the TGF-beta (transforming growth factor-beta)superfamily of proteins. In some embodiments, the atrogene comprisesDMPK. In some embodiments, the binding moiety is an antibody or bindingfragment thereof. In some embodiments, the binding moiety comprises ahumanized antibody or binding fragment thereof, chimeric antibody orbinding fragment thereof, monoclonal antibody or binding fragmentthereof, monovalent Fab′, divalent Fab2, single-chain variable fragment(scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), orcamelid antibody or binding fragment thereof. In some embodiments, thebinding moiety is an anti-transferrin receptor antibody or bindingfragment thereof. In some embodiments, the binding moiety ischolesterol. In some embodiments, the polynucleotide comprises a singlestrand which hybridizes to the target sequence of an atrogene. In someembodiments, the polynucleotide comprises a first polynucleotide and asecond polynucleotide hybridized to the first polynucleotide to form adouble-stranded polynucleic acid molecule, wherein either the firstpolynucleotide or the second polynucleotide also hybridizes to thetarget sequence of an atrogene. In some embodiments, the secondpolynucleotide comprises at least one modification. In some embodiments,the first polynucleotide and the second polynucleotide are RNAmolecules. In some embodiments, the polynucleotide hybridizes to atleast 8 contiguous bases of the target sequence of an atrogene. In someembodiments, the polynucleotide comprises a sequence that is at least60%, 70%, 80%, 85%, 90%, 95%, or 99% complementary to a sequence as setforth in SEQ ID NOs: 28-141, 370-480, and 703-3406. In some embodiments,the polynucleotide is between about 8 and about 50 nucleotides inlength. In some embodiments, the polynucleotide is between about 10 andabout 30 nucleotides in length. In some embodiments, the firstpolynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to a sequence as set forthin SEQ ID NOs: 142-255, 256-369, 481-591, 592-702, and 3407-14222. Insome embodiments, the second polynucleotide comprises a sequence havingat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a sequence as set forth in SEQ ID NOs: 142-255, 256-369,481-591, 592-702, and 3407-14222. In some embodiments, the polynucleicacid molecule conjugate optionally comprises a linker connecting thebinding moiety to the polynucleotide. In some embodiments, thepolynucleic acid molecule conjugate further comprises a polymer,optionally indirectly conjugated to the polynucleotide by an additionallinker. In some embodiments, the linker and the additional linker areeach independently a bond or a non-polymeric linker. In someembodiments, the polynucleic acid molecule conjugate comprises amolecule of Formula (I): A-X₁—B—X₂—C(Formula I) wherein, A is a bindingmoiety; B is a polynucleotide that hybridizes to a target sequence of anatrogene; C is a polymer; and X₁ and X₂ are each independently selectedfrom a bond or a non-polymeric linker; wherein the polynucleotidecomprises at least one 2′ modified nucleotide, at least one modifiedinternucleotide linkage, or at least one inverted abasic moiety; andwherein A and C are not attached to B at the same terminus. In someembodiments, the at least one 2′ modified nucleotide comprises2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified nucleotide. In some embodiments, the at least one 2′modified nucleotide comprises locked nucleic acid (LNA) or ethylenenucleic acid (ENA). In some embodiments, the at least one modifiedinternucleotide linkage comprises a phosphorothioate linkage or aphosphorodithioate linkage. In some embodiments, the at least oneinverted abasic moiety is at at least one terminus. In some embodiments,the muscle atrophy is a diabetes-associated muscle atrophy. In someembodiments, the muscle atrophy is a cancer cachexia-associated muscleatrophy. In some embodiments, the muscle atrophy is associated withinsulin deficiency. In some embodiments, the muscle atrophy isassociated with chronic renal failure. In some embodiments, the muscleatrophy is associated with congestive heart failure. In someembodiments, the muscle atrophy is associated with chronic respiratorydisease. In some embodiments, the muscle atrophy is associated with achronic infection. In some embodiments, the muscle atrophy is associatedwith fasting. In some embodiments, the muscle atrophy is associated withdenervation. In some embodiments, the muscle atrophy is associated withsarcopenia, glucocorticoid treatment, stroke, and/or heart attack. Insome cases, myotonic dystrophy type 1 (DM1) is associated with anexpansion of CTG repeats in the 3′ UTR of the DMPK gene.

Disclosed herein, in certain embodiments, is a pharmaceuticalcomposition comprising: a molecule described above or a polynucleic acidmolecule conjugate described above; and a pharmaceutically acceptableexcipient. In some embodiments, the pharmaceutical composition isformulated as a nanoparticle formulation. In some embodiments, thepharmaceutical composition is formulated for parenteral, oral,intranasal, buccal, rectal, or transdermal administration.

Disclosed herein, in certain embodiments, is a method of treating muscleatrophy or myotonic dystrophy in a subject in need thereof, comprising:administering to the subject a therapeutically effective amount of apolynucleic acid molecule conjugate comprising a binding moietyconjugated to a polynucleotide that hybridizes to a target sequence ofan atrogene; wherein the polynucleotide optionally comprises at leastone 2′ modified nucleotide, at least one modified internucleotidelinkage, or at least one inverted abasic moiety; and wherein thepolynucleic acid molecule conjugate mediates RNA interference againstthe atrogene, thereby treating muscle atrophy or myotonic dystrophy inthe subject. In some embodiments, the muscle atrophy is adiabetes-associated muscle atrophy. In some embodiments, the muscleatrophy is a cancer cachexia-associated muscle atrophy. In someembodiments, the muscle atrophy is associated with insulin deficiency.In some embodiments, the muscle atrophy is associated with chronic renalfailure. In some embodiments, the muscle atrophy is associated withcongestive heart failure. In some embodiments, the muscle atrophy isassociated with chronic respiratory disease. In some embodiments, themuscle atrophy is associated with a chronic infection. In someembodiments, the muscle atrophy is associated with fasting. In someembodiments, the muscle atrophy is associated with denervation. In someembodiments, the muscle atrophy is associated with sarcopenia. In someembodiments, the myotonic dystrophy is DM1. In some embodiments, theatrogene comprises a differently regulated (e.g., an upregulated ordownregulated) gene within the IGF1-Akt-FoxO pathway, theglucocorticoids-GR pathway, the PGC1α-FoxO pathway, the TNFα-NFκBpathway, or the myostatin-ActRIIb-Smad2/3 pathway. In some embodiments,the atrogene encodes an E3 ligase. In some embodiments, the atrogeneencodes a Forkhead box transcription factor. In some embodiments, theatrogene comprises atrogin-1 gene (FBXO32), MuRF1 gene (TRIM63), FOXO1,FOXO3, or MSTN. In some embodiments, the atrogene comprises DMPK. Insome embodiments, the polynucleic acid molecule conjugate comprises amolecule of Formula (I): A-X₁—B—X₂—C(Formula I) wherein, A is a bindingmoiety; B is a polynucleotide that hybridizes to the target sequence ofan atrogene; C is a polymer; and X₁ and X₂ are each independentlyselected from a bond or a non-polymeric linker; wherein thepolynucleotide comprises at least one 2′ modified nucleotide, at leastone modified internucleotide linkage, or at least one inverted abasicmoiety; and wherein A and C are not attached to B at the same terminus.In some embodiments, B consists of a polynucleotide that hybridizes tothe target sequence of an atrogene. In some embodiments, C consists of apolymer. In some embodiments, the at least one 2′ modified nucleotidecomprises 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl,2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA) modified nucleotide. In someembodiments, the at least one 2′ modified nucleotide comprises lockednucleic acid (LNA) or ethylene nucleic acid (ENA). In some embodiments,the at least one modified internucleotide linkage comprises aphosphorothioate linkage or a phosphorodithioate linkage. In someembodiments, the at least one inverted abasic moiety is at at least oneterminus. In some embodiments, the polynucleotide comprises a singlestrand which hybridizes to the target sequence of an atrogene. In someembodiments, the polynucleotide comprises a first polynucleotide and asecond polynucleotide hybridized to the first polynucleotide to form adouble-stranded polynucleic acid molecule, wherein either the firstpolynucleotide or the second polynucleotide also hybridizes to thetarget sequence of an atrogene. In some embodiments, the secondpolynucleotide comprises at least one modification. In some embodiments,the first polynucleotide and the second polynucleotide are RNAmolecules. In some embodiments, the polynucleotide hybridizes to atleast 8 contiguous bases of the target sequence of an atrogene. In someembodiments, the polynucleotide comprises a sequence that is at least60%, 70%, 80%, 85%, 90%, 95%, or 99% complementary to a sequence as setforth in SEQ ID NOs: 28-141, 370-480, and 703-3406. In some embodiments,the polynucleotide is between about 8 and about 50 nucleotides inlength. In some embodiments, the polynucleotide is between about 10 andabout 30 nucleotides in length. In some embodiments, the firstpolynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to a sequence as set forthin SEQ ID NOs: 142-255, 256-369, 481-591, 592-702, and 3407-14222. Insome embodiments, the second polynucleotide comprises a sequence havingat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a sequence as set forth in SEQ ID NOs: 142-255, 256-369,481-591, 592-702, and 3407-14222. In some embodiments, X₁ and X₂ areindependently a C₁-C₆ alkyl group. In some embodiments, X₁ and X₂ areindependently a homobifuctional linker or a heterobifunctional linker,optionally conjugated to a C₁-C₆ alkyl group. In some embodiments, A isan antibody or binding fragment thereof. In some embodiments, Acomprises a humanized antibody or binding fragment thereof, chimericantibody or binding fragment thereof, monoclonal antibody or bindingfragment thereof, monovalent Fab′, divalent Fab2, single-chain variablefragment (scFv), diabody, minibody, nanobody, single-domain antibody(sdAb), or camelid antibody or binding fragment thereof. In someembodiments, A is an anti-transferrin receptor antibody or bindingfragment thereof. In some embodiments, C is polyethylene glycol. In someembodiments, A-X₁ is conjugated to the 5′ end of B and X₂—C isconjugated to the 3′ end of B. In some embodiments, X₂—C is conjugatedto the 5′ end of B and A-X₁ is conjugated to the 3′ end of B. In someembodiments, A is directly conjugated to X₁. In some embodiments, C isdirectly conjugated to X₂. In some embodiments, B is directly conjugatedto X₁ and X₂. In some embodiments, the method further comprises D. Insome embodiments, D is conjugated to C or to A. In some embodiments, Dis an endosomolytic polymer. In some embodiments, the polynucleic acidmolecule conjugate is formulated for parenteral, oral, intranasal,buccal, rectal, or transdermal administration. In some embodiments, thesubject is a human.

Disclosed herein, in certain embodiments, is a kit comprising a moleculedescribed above or a polynucleic acid molecule conjugate describedabove.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings below. The patent application file contains atleast one drawing executed in color. Copies of this patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

FIG. 1 illustrates an exemplary structure of cholesterol-myostatin siRNAconjugate.

FIG. 2 illustrates SAX HPLC chromatogram of TfR mAb-(Cys)-HPRT-PEG5k,DAR1.

FIG. 3 illustrates SEC HPLC chromatogram of TfR mAb-(Cys)-HPRT-PEG5k,DAR1.

FIG. 4 illustrates an overlay of DAR1 and DAR2 SAX HPLC chromatograms ofTfR1mAb-Cys-BisMal-siRNA conjugates.

FIG. 5 illustrates an overlay of DAR1 and DAR2 SEC HPLC chromatograms ofTfR1mAb-Cys-BisMal-siRNA conjugates.

FIG. 6 illustrates SEC chromatogram of CD71 Fab-Cys-HPRT-PEG5.

FIG. 7 illustrates SAX chromatogram of CD71 Fab-Cys-HPRT-PEG5.

FIG. 8 illustrates relative expression levels of Murf1 and atrogin-1 inC2C12 myoblasts and myotubes C2C12 myoblasts and myotubes were generatedas described in Example 4. mRNA levels were determined as described inExample 4.

FIG. 9A illustrates in vivo study design to assess the ability ofexemplary conjugates for their ability to mediate mRNA downregulation ofmyostatin (MSTN) in skeletal muscle.

FIG. 9B shows siRNA-mediated mRNA knockdown of mouse MSTN in mousegastrocnemius (gastroc) muscle.

FIG. 10A illustrates in vivo study design to assess the ability ofexemplary conjugates for their ability to mediate mRNA downregulation ofmyostatin (MSTN) in skeletal muscle.

FIG. 10B shows tissue concentration-time profiles out to 1008 hpost-dose of an exemplary molecule of Formula (I).

FIG. 10C shows siRNA-mediated mRNA knockdown of mouse MSTN in mousegastrocnemius (gastroc) muscle.

FIG. 10D shows plasma MSTN protein reduction after siRNA-mediated mRNAknockdown of mouse MSTN in mouse gastrocnemius (gastroc) muscle.

FIG. 10E shows changes in muscle size after siRNA-mediated mRNAknockdown of mouse MSTN in mouse gastrocnemius (gastroc) muscle.

FIG. 10F shows Welch's two-tailed unpaired t-test of FIG. 10E.

FIG. 11A illustrates an exemplary in vivo study design.

FIG. 11B shows tissue accumulation of siRNA in mouse gastrocnemius(gastroc) muscle after a single i.v. administration of an exemplarymolecule of Formula (I) at the doses indicated.

FIG. 11C shows siRNA-mediated mRNA knockdown of mouse MSTN in mousegastrocnemius (gastroc) muscle.

FIG. 12A illustrates an exemplary in vivo study design.

FIG. 12B shows accumulation of siRNA in various muscle tissue.

FIG. 12C shows siRNA-mediated mRNA knockdown of mouse MSTN in mousegastrocnemius (gastroc) and heart muscle.

FIG. 12D shows RISC loading of the MSTN guide strand in mousegastrocnemius (gastroc) muscle.

FIG. 13A illustrates an exemplary in vivo study design.

FIG. 13B shows siRNA-mediated mRNA knockdown of mouse MSTN in mousegastrocnemius (gastroc), quadriceps, triceps, and heart.

FIG. 13C illustrates plasma myostatin levels.

FIG. 13D illustrates siRNA accumulation in different tissue types:gastrocnemius, triceps, quadriceps, and heart tissues.

FIG. 13E shows RISC loading of the MSTN guide strand in mousegastrocnemius (gastroc) muscle.

FIG. 13F shows change in muscle area.

FIG. 13G shows Welch's two-tailed unpaired t-test of FIG. 13F.

FIG. 14A illustrates an exemplary in vivo study design.

FIG. 14B shows HPRT mRNA expression of gastrocnemius muscle by exemplaryconjugates described herein.

FIG. 14C shows SSB mRNA expression of gastrocnemius muscle by exemplaryconjugates described herein.

FIG. 14D shows HPRT mRNA expression of heart tissue by exemplaryconjugates described herein.

FIG. 14E shows SSB mRNA expression of heart tissue by exemplaryconjugates described herein.

FIG. 14F shows accumulation of siRNA in gastrocnemius muscle.

FIG. 15A illustrates an exemplary in vivo study design.

FIG. 15B shows Atrogin-1 downregulation in gastrocnemius (gastroc)muscle.

FIG. 15C shows Atrogin-1 downregulation in heart tissue.

FIG. 16A illustrates an exemplary in vivo study design.

FIG. 16B shows MuRF-1 downregulation in gastrocnemius muscle.

FIG. 16C shows MuRF-1 downregulation in heart tissue.

FIG. 17 illustrates siRNAs that were transfected into mouse C2C12myoblasts in vitro. The four DMPK siRNAs assessed all showed DMPK mRNAknockdown, while the negative control siRNA did not. The dotted linesare three-parameter curves fit by non-linear regression.

FIG. 18A-FIG. 18F show in vivo results demonstrating robustdose-responses for DMPK mRNA knockdown 7 days after a single i.v.administration of DMPK siRNA-antibody conjugates. FIG. 18A:gastrocnemius; FIG. 18B: Tibialis anterior; FIG. 18C: quadriceps; FIG.18D: diaphragm; FIG. 18E: heart; and FIG. 18F: liver.

FIG. 19A-FIG. 19L show exemplary antibody-nucleic acid conjugatesdescribed herein.

FIG. 19M presents an antibody cartoon utilized in FIG. 19A-FIG. 19L.

FIG. 20A-FIG. 20B illustrate an exemplary 21mer duplex utilized inExample 20. FIG. 20A shows a representative structure of siRNA passengerstrand with C6-NH₂ conjugation handle at the 5′ end and C6-S-NEM at 3′end. FIG. 20B shows a representative structure of a 21mer duplex with 19bases of complementarity and 3′ dinucleotide overhangs.

FIG. 21A-FIG. 21B illustrate a second exemplary 21 mer duplex utilizedin Example 20. FIG. 21A shows a representative structure of siRNApassenger strand with a 5′ conjugation handle. FIG. 21B shows arepresentative structure of a blunt ended duplex with 19 bases ofcomplementarity and one 3′ dinucleotide overhang.

FIG. 22 shows an illustrative in vivo study design.

FIG. 23 illustrates a time course of Atrogin-1 mRNA downregulation ingastroc muscle mediated by a TfR1 antibody siRNA conjugate after IVdelivery at a dose of a single dose of 3 mg/kg.

FIG. 24 illustrates a time course of Atrogin-1 mRNA downregulation inheart muscle mediate by a TfR1 antibody siRNA conjugate after IVdelivery at a dose of a single dose of 3 mg/kg.

FIG. 25 shows an illustrative in vivo study design.

FIG. 26 shows MuRF1 mRNA downregulation at 96 hours in gastroc musclemediated by a TfR1 antibody siRNA conjugate after IV delivery at thedoses indicated.

FIG. 27 shows MuRF1 mRNA downregulation at 96 hours in heart musclemediated by a TfR1 antibody siRNA conjugate after IV delivery at thedoses indicated.

FIG. 28 shows a time course of MuRF1 and Atrogin-1 mRNA downregulationin gastroc muscle mediated by a TfR1 antibody siRNA conjugate (IVdelivery at 3 mg/kg siRNA), in the absence and presence of dexamethasoneinduce muscle atrophy.

FIG. 29 shows a time course of MuRF1 and Agtrogin1 mRNA downregulationin heart muscle mediated by a TfR1 antibody siRNA conjugate (IV deliveryat 3 mg/kg siRNA), in the absence and presence of dexamethasone inducemuscle atrophy.

FIG. 30 shows a time course of gastroc weight changes mediated by a TfR1antibody siRNA conjugate (IV delivery at 3 mg/kg siRNA), in the absenceand presence of muscle atrophy.

FIG. 31 shows a time course of siRNA tissue concentrations in gastrocand heart muscle mediated by a TfR1 antibody siRNA conjugate (IVdelivery at 3 mg/kg siRNA), in the absence and presence of muscleatrophy.

FIG. 32 shows an illustrative in vivo study design.

FIG. 33 shows Atrogin-1 mRNA downregulation in gastroc muscle, 10 daysafter TfR1 antibody siRNA conjugate, in the absence a presence ofdexamethasone induced atrophy (initiated at day 7), relative to themeasure concentration of siRNA in the tissue.

FIG. 34 shows relative Atrogin-1 mRNA levels in gastroc muscle for thescrambled control groups in the absence (groups 10&13, and groups 11l&14)) and presence of dexamethasone induced atrophy (groups 12&15).

FIG. 35 shows relative RISC loading of the Atrogin-1 guide strand inmouse gastroc muscle after TfR1-mAb conjugate delivery in the absenceand presence of dexamethasone induced atrophy.

FIG. 36 shows a time course of MSTN mRNA downregulation in gastrocmuscle after TfR1 antibody siRNA conjugate delivery, in the absence(solid lines) and presence (dotted lines) of dexamethasone inducedatrophy (initiated at day 7), relative to the PBS control.

FIG. 37 shows leg muscle growth rate in gastroc muscle, after TfR1-mAbconjugate delivery in the absence and presence of dexamethasone inducedatrophy.

FIG. 38 shows an illustrative in vivo study design.

FIG. 39A shows a single treatment of 4.5 mg/kg (siRNA) of eitherAtrogin-1 siRNA or MuRFI siRNA or a single dose of both siRNAs combinedresulted in up to 75% downregulation of each target in thegastrocnemius.

FIG. 39B shows mRNA knockdown of both targets in gastrocnemius ismaintained at 75% in the intact leg out to 37 days post ASC dose.

FIG. 39C shows changes in muscle area.

FIG. 39D shows changes in gastrocnemius weight.

FIG. 39E shows treatment-induced percentage sparing of muscle wasting interm of leg muscle area. The statistical analysis compared the treatmentgroups to the scramble siRNA control group using a Welch's TTest.

FIG. 39F shows the treatment-induced percentage sparing of musclewasting in term of gastrocnemius weight.

FIG. 40A shows a representative structure of siRNA with C6-NH₂conjugation handle at the 5′ end and C6-SH at 3′end of the passengerstrand.

FIG. 40B shows a representative structure of siRNA passenger strand withC6-NH₂ conjugation handle at the 5′ end and C6-S-PEG at 3′ end.

FIG. 40C shows a representative structure of siRNA passenger strand withC6-NH₂ conjugation handle at the 5′ end and C6-S-NEM at 3′ end.

FIG. 40D shows a representative structure of siRNA passenger strand withC6-N-SMCC conjugation handle at the 5′ end and C6-S-NEM at 3′ end.

FIG. 40E shows a representative structure of siRNA passenger strand withPEG at the 5′ end and C6-SH at 3′ end.

FIG. 40F shows a representative structure of siRNA passenger strand withC6-S-NEM at the 5′ end and C6-NH₂ conjugation handle at 3′ end.

FIG. 41A shows Architecture-1: Antibody-Cys-SMCC-5′-passenger strand.This conjugate was generated by antibody inter-chain cysteineconjugation to maleimide (SMCC) at the 5′ end of passenger strand.

FIG. 41B shows Architecture-2: Antibody-Cys-SMCC-3′-Passenger strand.This conjugate was generated by antibody inter-chain cysteineconjugation to maleimide (SMCC) at the 3′ end of passenger strand.

FIG. 41C shows ASC Architecture-3: Antibody-Cys-bisMal-3′-Passengerstrand. This conjugate was generated by antibody inter-chain cysteineconjugation to bismaleimide (bisMal) linker at the 3′ end of passengerstrand.

FIG. 41D shows ASC Architecture-4: A model structure of theFab-Cys-bisMal-3′-Passenger strand. This conjugate was generated by Fabinter-chain cysteine conjugation to bismaleimide (bisMal) linker at the3′ end of passenger strand.

FIG. 41E shows ASC Architecture-5: A model structure of the antibodysiRNA conjugate with two different siRNAs attached to one antibodymolecule. This conjugate was generated by conjugating a mixture of SSBand HPRT siRNAs to the reduced mAb inter-chain cysteines to bismaleimide(bisMal) linker at the 3′ end of passenger strand of each siRNA.

FIG. 41F shows ASC Architecture-6: A model structure of the antibodysiRNA conjugate with two different siRNAs attached. This conjugate wasgenerated by conjugating a mixture of SSB and HPRT siRNAs to the reducedmAb inter-chain cysteines to maleimide (SMCC) linker at the 3′ end ofpassenger strand of each siRNA.

FIG. 42 shows Synthesis scheme-1: Antibody-Cys-SMCC-siRNA-PEG conjugatesvia antibody cysteine conjugation.

FIG. 43 shows Synthesis scheme-2: Antibody-Cys-BisMal-siRNA-PEGconjugates.

FIG. 44 shows Scheme-3: Fab-siRNA conjugate generation.

DETAILED DESCRIPTION OF THE DISCLOSURE

Muscle atrophy is the loss of muscle mass or the progressive weakeningand degeneration of muscles, such as skeletal or voluntary muscles thatcontrols movement, cardiac muscles, and smooth muscles. Variouspathophysiological conditions including disuse, starvation, cancer,diabetes, and renal failure, or treatment with glucocorticoids result inmuscle atrophy and loss of strength. The phenotypical effects of muscleatrophy are induced by various molecular events, including inhibition ofmuscle protein synthesis, enhanced turnover of muscle proteins, abnormalregulation of satellite cells differentiation, and abnormal conversionof muscle fibers types.

Extensive research has identified that muscle atrophy is an activeprocess controlled by specific signaling pathways and transcriptionalprograms. Exemplary pathways involved in this process include, but arenot limited to, IGF1-Akt-FoxO, glucocorticoids-GR, PGC1α-FoxO,TNFα-NFκB, and myostatin-ActRIIb-Smad2/3.

In some instances, therapeutic manipulation of mechanisms regulatingmuscle atrophy has focused on IGF1-Akt, TNFα-NfκB, and myostatin. WhileIGF1 analogs were shown to be effective in treating muscle atrophy, theinvolvement of the IGF1-Akt pathway in promoting tumorigenesis andhypertrophy prevents these therapies. Similar risks are involved in theuse of $3-adrenergic agonists for the regulation of the Akt-mTORpathway. Inhibition of myostatin by using soluble ActRIIB or ligandblocking ActRIIb antibodies prevented and reversed skeletal muscle loss,and prolonged the survival of tumor-bearing animals. However themechanism of the anti-atrophic effects of myostatin blockade remainsuncertain as neither expression of a dominant-negative ActRIIb, norknockdown of Smad2/3 prevented muscle loss following denervation (Satoriet al., “Smad2 and 3 transcription factors control muscle mass inadulthood”, Am J Physiol Cell Physiol 296: C1248-C1257, 2009).

Comparing gene expression in different models of muscle atrophy(including diabetes, cancer cachexia, chronic renal failure, fasting anddenervation) has led to the identification of atrophy-related genes,named atrogenes (Sacheck et al., “Rapid disuse and denervation atrophyinvolve transcriptional changes similar to those of muscle wastingduring systemic diseases”, The FASEB Journal, 21(1): 140-155, 2007),that are commonly up- or downregulated in atrophying muscle. Among genesthat are strongly upregulated under atrophy conditions aremuscle-specific ubiquitin-protein (E3) ligases (e.g. atrogin-1, MuRF1),Forkhead box transcription factors, and proteins mediating stressresponses. In some cases, many of these effector proteins are difficultto regulate using traditional drugs.

Nucleic acid (e.g., RNAi) therapy is a targeted therapy with highselectivity and specificity. However, in some instances, nucleic acidtherapy is also hindered by poor intracellular uptake, limited bloodstability and non-specific immune stimulation. To address these issues,various modifications of the nucleic acid composition are explored, suchas for example, novel linkers for better stabilizing and/or lowertoxicity, optimization of binding moiety for increased targetspecificity and/or target delivery, and nucleic acid polymermodifications for increased stability and/or reduced off-target effect.

In some embodiments, the arrangement or order of the differentcomponents that make-up the nucleic acid composition further effectsintracellular uptake, stability, toxicity, efficacy, and/or non-specificimmune stimulation. For example, if the nucleic acid component includesa binding moiety, a polymer, and a polynucleic acid molecule (orpolynucleotide), the order or arrangement of the binding moiety, thepolymer, and/or the polynucleic acid molecule (or polynucleotide) (e.g.,binding moiety-polynucleic acid molecule-polymer, bindingmoiety-polymer-polynucleic acid molecule, or polymer-bindingmoiety-polynucleic acid molecule) further effects intracellular uptake,stability, toxicity, efficacy, and/or non-specific immune stimulation.

In some embodiments, described herein include polynucleic acid moleculesand polynucleic acid molecule conjugates for the treatment of muscleatrophy or myotonic dystrophy. In some instances, the polynucleic acidmolecule conjugates described herein enhance intracellular uptake,stability, and/or efficacy. In some cases, the polynucleic acid moleculeconjugates comprise a molecule of Formula (I): A-X—B—X₂—C. In somecases, the polynucleic acid molecules that hybridize to target sequencesof one or more atrogenes.

Additional embodiments described herein include methods of treatingmuscle atrophy or myotonic dystrophy, comprising administering to asubject a polynucleic acid molecule or a polynucleic acid moleculeconjugate described herein.

Atrogenes

Atrogenes, or atrophy-related genes, are genes that are upregulated ordownregulated in atrophying muscle. In some instances, upregulatedatrogenes include genes that encode ubiquitin ligases, Forkhead boxtranscription factors, growth factors, deubiquitinating enzymes, orproteins that are involved in glucocorticoid-induced atrophy.

Ubiquitin Ligases

In some embodiments, an atrogene described herein encodes an E3ubiquitin ligase. Exemplary E3 ubiquitin ligases include, but are notlimited to, Atrogin-1/MAFbx, muscle RING finger 1 (MuRF1), TNF receptoradaptor protein 6 (TRAF6), F-Box protein 30 (Fbxo30), F-Box protein 40(Fbxo40), neural precursor cell expressed developmentally down-regulatedprotein 4 (Nedd4-1), and tripartite motif-containing protein 32(Trim32). Exemplary mitochondrial ubiquitin ligases include, but are notlimited to, Mitochondrial E3 ubiquitin protein ligase 1 (Mull) andCarboxy terminus of Hsc70 interacting protein (CHIP).

In some embodiments, an atrogene described herein encodes Atrogin-1,also named Muscle Atrophy F-box (MAFbx), a member of the F-box proteinfamily. Atrogin-1/MAFbx is one of the four subunits of the ubiquitinligase complex SKP1-cullin-F-box (SCF) that promotes degradation ofMyoD, a muscle transcription factor, and eukaryotic translationinitiation factor 3 subunit F (eIF3-f). Atrogin-1/MAFbx is encoded byFBXO32.

In some embodiments, an atrogene described herein encodes muscle RINGfinger 1 (MuRF1). MuRF1 is a member of the muscle-specific RING fingerproteins and along with family members MuRF2 and MuRF3 are found at theM-line and Z-line lattices of myofibrils. Further, several studies haveshown that MuRF1 interacts with and/or modulates the half-life of musclestructural proteins such as troponin I, myosin heavy chains, actin,myosin binding protein C, and myosin light chains 1 and 2. MuRF1 isencoded by TRIM63.

In some embodiments, an atrogene described herein encodes TNF receptoradaptor protein 6 (TRAF6) (also known as interleukin-1 signaltransducer, RING finger protein 85, or RNF85). TRAF6 is a member of theE3 ligase that mediates conjugation of Lys63-linked polyubiquitin chainsto target proteins. The Lys63-linked polyubiquitin chains signalautophagy-dependent cargo recognition by scaffold protein p62 (SQSTM1).TRAF6 is encoded by the TRAF6 gene.

In some embodiments, an atrogene described herein encodes F-Box protein30 (Fbxo30) (also known as F-Box only protein, helicase, 18; muscleubiquitin ligase of SCF complex in atrophy-1; or MUSA1). Fbxo30 is amember of the SCF complex family of E3 ubiquitin ligases. In one study,Fbox30 is proposed to be inhibited by the bone morphogenetic protein(BMP) pathway and upon atrophy-inducing conditions, are upregulated andsubsequently undergoes autoubiquitination. Fbxo30 is encoded by theFBXO30 gene.

In some embodiments, an atrogene described herein encodes F-Box protein40 (Fbxo40) (also known as F-Box only protein 40 or muscledisease-related protein). A second member of the SCF complex family ofE3 ubiquitin ligases, Fbxo40 regulates anabolic signals. In someinstances, Fbxo40 ubiquitinates and affects the degradation of insulinreceptor substrate 1, a downstream effector of insulin receptor-mediatedsignaling. Fbxo40 is encoded by the FBXO40 gene.

In some embodiments, an atrogene described herein encodes neuralprecursor cell expressed developmentally down-regulated protein 4(Nedd4-1), a HECT domain E3 ubiquitin ligase which has been shown to beupregulated in muscle cells during disuse. Nedd4-1 is encoded by theNEDD4 gene.

In some embodiments, an atrogene described herein encodes tripartitemotif-containing protein 32 (Trim32). Trim32 is a member of the E3ubiquitin ligase that is involved in degradation of thin filaments suchas actin, tropomyosin, and troponins; α-actinin; and desmin. Trim32 isencoded by the TRIM32 gene.

In some embodiments, an atrogene described herein encodes MitochondrialE3 ubiquitin protein ligase 1 (Mull) (also known asmitochondrial-anchored protein ligase, RING finger protein 218, RNF218,MAPL, MULAN, and GIDE). Mull is involved in the mitochondrial networkremodeling and is up-regulated by the FoxO family of transcriptionfactors under catabolic conditions, such as for example, denervation orfasting, and subsequently causes mitochondrial fragmentation and removalvia autophagy (mitophagy). Furthermore, Mull ubiquitinates themitochondrial pro-fusion protein mitofusin 2, a GTPase that is involvedin mitochondrial fusion, leading to the degradation of mitofusin 2. Mullis encoded by the MUL1 gene.

In some embodiments, an atrogene described herein encodes Carboxyterminus of Hsc70 interacting protein (CHIP) (also known as STIP1homology and U-Box containing protein 1, STUB1, CLL-associated antigenKW-8, antigen NY—CO-7, SCAR16, SDCCAG7, or UBOX1). CHIP is amitochondrial ubiquitin ligase that regulates ubiquitination andlysosomal-dependent degradation of filamin C, a muscle protein found inthe Z-line. Z-line or Z-disc is the structure formed between adjacentsarcomeres, and sarcomere is the basic unit of muscle. Alterations offilamin structure triggers binding of the co-chaperone BAG3, a complexthat comprises chaperones Hsc70 and HspB8 with CHIP. Subsequentubiquitination of BAG3 and filamin by CHIP activates the autophagysystem, leading to degradation of filamin C. CHIP is encoded by theSTUB1 gene.

Forkhead Box Transcription Factors

In some embodiments, an atrogene described herein encodes a Forkhead boxtranscription factor. Exemplary Forkhead box transcription factorsinclude, but are not limited to, isoforms Forkhead box protein 01(FoxO1) and Forkhead box protein O3 (FoxO3).

In some embodiments, an atrogene described herein encodes Forkhead boxprotein 01 (FoxO1) (also known as Forkhead homolog in Rhabdomyoscarcoma,FKHR, or FKH1). FoxO1 is involved in regulation of gluconeogenesis andglycogenolysis by insulin signaling, and the initiation of adipogenesisby preadipocytes. FoxO1 is encoded by the FOXO1 gene.

In some embodiments, an atrogene described herein encodes Forkhead boxprotein 03 (FoxO3) (also known as Forkhead in Rhabdomyosarcoma-like 1,FKHRL1, or FOXO3A). FoxO3 is activated by AMP-activated protein kinaseAMPK, which in term induces expression of atrogin-1 and MuRF1. FoxO3 isencoded by the FOXO3 gene.

Growth Factors

In some embodiments, an atrogene described herein encodes a growthfactor. An exemplary growth factor includes myostatin.

In some instances, an atrogene described herein encodes myostatin(Mstn), also known as growth/differentiation factor 8 (GDF-8). Myostatinis intracellularly converted into an activator, and stimulates muscledegradation and suppresses muscle synthesis by inhibiting Akt throughthe phosphorylation/activation of Smad (small mothers againstdecapentaplegic). In some instances, myostatin has been found to beregulated by the Akt-FoxO signaling pathway. In additional instances,myostatin has been shown to suppress differentiation of satellite cells,stimulate muscle degradation through the inhibition of the Akt pathway,and suppress muscle synthesis via the mTOR pathway.

Deubiquitinating Enzymes

In some embodiments, an atrogene described herein encodes adeubiquitinating enzyme. Exemplary deubiquitinating enzymes include, butare not limited to, Ubiquitin specific peptidase 14 (USP14) andUbiquitin specific peptidase 19 (USP19). In some instances, an atrogenedescribed herein encodes USP14 (also known as deubiquitinating enzyme 14or TGT). In other instances, an atrogene described herein encodes USP19(also known as zinc finger MYND domain-containing protein 9,deubiquitinating enzyme 19, or ZMYND9). USP14 is encoded by the USP14gene. USP19 is encoded by the USP19 gene.

Additional Atrogenes

In some embodiments, an atrogene described herein encodes regulated indevelopment and DNA damage response 1 (Redd1), also known asDNA-damage-inducible transcript 4 (DDIT4) and HIF-1 responsive proteinRTP801. Redd1 represses mTOR function by sequestering 14-3-3 andincreases TSC1/2 activity. Furthermore, Redd1 decreases phosphorylationof 4E-BP1 and S6K1, which are involved in muscle protein synthesis.Redd1 is encoded by the DDIT4 gene.

In some embodiments, an atrogene described herein encodes cathepsin L2,also known as cathepsin V. Cathepsin L2 is a lysosomal cysteineproteinase. It is encoded by the CTSL2 gene.

In some embodiments, an atrogene described herein encodes TG interactingfactor, or homeobox protein TGIF1. TG interacting factor is atranscription factor which regulates signaling pathways involved inembryonic development. This protein is encoded by the TGIF gene.

In some embodiments, an atrogene described herein encodes myogenin, alsoknown as myogenic factor 4. Myogenin is a member of the MyoD family ofmuscle-specific basic-helix-loop-helix (bHLH) transcription factorinvolved in the coordination of skeletal muscle development and repair.Myogenin is encoded by the MYOG gene.

In some embodiments, an atrogene described herein encodesmyotonin-protein kinase (MT-PK), also known as myotonic dystrophyprotein kinase (MDPK) or dystrophia myotonica protein kinase (DMK).MT-PK is a Serine/Threonine kinase and further interacts with members ofthe Rho family of GTPases. In human, MT-PK is encoded by the DMPK gene.

In some embodiments, an atrogene described herein encodes histonedeacetylase 2, a member of the histone deacetylase family. Histonedeacetylase 2 is encoded by the HDAC2 gene.

In some embodiments, an atrogene described herein encodes histonedeacetylase 3, another member of the histone deacetylase family. Histonedeacetylase 3 is encoded by the HDAC3 gene.

In some embodiments, an atrogene described herein encodesmetallothionein 1L, a member of the metallothionein family.Metallothioneins (MT) are cysteine-rish, low molecular weight proteinsthat is capable of binding heavy metals, thereby providing protectionagainst metal toxicity and/or oxidative stress. Metallothionein 1L isencoded by the MT1L gene.

In some embodiments, an atrogene described herein encodesmetallothionein 1B, a second member of the metallothionein family.Metallothionein 1B is encoded by the MT1B gene.

In some embodiments, an atrogene described herein is an atrogene listedin Table 14.

Polynucleic Acid Molecules

In certain embodiments, a polynucleic acid molecule hybridizes to atarget sequence of an atrophy-related gene (also referred to as anatrogene). In some instances, a polynucleic acid molecule describedherein hybridizes to a target sequence of an ubiquitin ligase (e.g., anE3 ubiquitin ligase or a mitochondrial ubiquitin ligase). In someinstances, a polynucleic acid molecule described herein hybridizes to atarget sequence of a Forkhead box transcription factor. In someinstances, a polynucleic acid molecule described herein hybridizes to atarget sequence of a growth factor. In some instances, a polynucleicacid molecule described herein hybridizes to a target sequence of adeubiquitinating enzyme.

In some embodiments, a polynucleic acid molecule described hereinhybridizes to a target sequence of FBXO32, TRIM63, TRAF6, FBXO30,FBXO40, NEDD4, TRIM32, MUL1, STUB1, FOXO1, FOXO3, MSTN, USP14, USP19,DDIT4, CTSL2, TGIF, MYOG, HDAC2, HDA6C3, MT1L, MT1B, or DMPK. In somecases, a polynucleic acid molecule described herein hybridizes to atarget sequence of FBXO32, TRIM63, FOXO1, FOXO3, or MSTN. In some cases,a polynucleic acid molecule described herein hybridizes to a targetsequence of FBXO32. In some cases, a polynucleic acid molecule describedherein hybridizes to a target sequence of TRIM63. In some cases, apolynucleic acid molecule described herein hybridizes to a targetsequence of TRAF6. In some cases, a polynucleic acid molecule describedherein hybridizes to a target sequence of FBXO30. In some cases, apolynucleic acid molecule described herein hybridizes to a targetsequence of FBXO40. In some cases, a polynucleic acid molecule describedherein hybridizes to a target sequence of NEDD4. In some cases, apolynucleic acid molecule described herein hybridizes to a targetsequence of TRIM32. In some cases, a polynucleic acid molecule describedherein hybridizes to a target sequence of MUL1. In some cases, apolynucleic acid molecule described herein hybridizes to a targetsequence of STUB1. In some cases, a polynucleic acid molecule describedherein hybridizes to a target sequence of FOXO1. In some cases, apolynucleic acid molecule described herein hybridizes to a targetsequence of FOXO3. In some cases, a polynucleic acid molecule describedherein hybridizes to a target sequence of MSTN. In some cases, apolynucleic acid molecule described herein hybridizes to a targetsequence of USP14. In some cases, a polynucleic acid molecule describedherein hybridizes to a target sequence of USP19. In some cases, apolynucleic acid molecule described herein hybridizes to a targetsequence of DDIT4. In some cases, a polynucleic acid molecule describedherein hybridizes to a target sequence of CTSL2. In some cases, apolynucleic acid molecule described herein hybridizes to a targetsequence of TGIF. In some cases, a polynucleic acid molecule describedherein hybridizes to a target sequence of MYOG. In some cases, apolynucleic acid molecule described herein hybridizes to a targetsequence of HDAC2. In some cases, a polynucleic acid molecule describedherein hybridizes to a target sequence of HDAC3. In some cases, apolynucleic acid molecule described herein hybridizes to a targetsequence of MT1L. In some cases, a polynucleic acid molecule describedherein hybridizes to a target sequence of MT1B. In some cases, apolynucleic acid molecule described herein hybridizes to a targetsequence of of DMPK.

In some embodiments, the polynucleic acid molecule comprises a sequencehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to a target sequence as setforth in SEQ ID NOs: 28-141 and 370-480. In some embodiments, thepolynucleic acid molecule comprises a sequence having at least 50%sequence identity to a target sequence as set forth in SEQ ID NOs:28-141 and 370-480. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 60% sequence identity to a targetsequence as set forth in SEQ ID NOs: 28-141 and 370-480. In someembodiments, the polynucleic acid molecule comprises a sequence havingat least 70% sequence identity to a target sequence as set forth in SEQID NOs: 28-141 and 370-480. In some embodiments, the polynucleic acidmolecule comprises a sequence having at least 75% sequence identity to atarget sequence as set forth in SEQ ID NOs: 28-141 and 370-480. In someembodiments, the polynucleic acid molecule comprises a sequence havingat least 80% sequence identity to a target sequence as set forth in SEQID NOs: 28-141 and 370-480. In some embodiments, the polynucleic acidmolecule comprises a sequence having at least 85% sequence identity to atarget sequence as set forth in SEQ ID NOs: 28-141 and 370-480. In someembodiments, the polynucleic acid molecule comprises a sequence havingat least 90% sequence identity to a target sequence as set forth in SEQID NOs: 28-141 and 370-480. In some embodiments, the polynucleic acidmolecule comprises a sequence having at least 95% sequence identity to atarget sequence as set forth in SEQ ID NOs: 28-141 and 370-480. In someembodiments, the polynucleic acid molecule comprises a sequence havingat least 96% sequence identity to a target sequence as set forth in SEQID NOs: 28-141 and 370-480. In some embodiments, the polynucleic acidmolecule comprises a sequence having at least 97% sequence identity to atarget sequence as set forth in SEQ ID NOs: 28-141 and 370-480. In someembodiments, the polynucleic acid molecule comprises a sequence havingat least 98% sequence identity to a target sequence as set forth in SEQID NOs: 28-141 and 370-480. In some embodiments, the polynucleic acidmolecule comprises a sequence having at least 99% sequence identity to atarget sequence as set forth in SEQ ID NOs: 28-141 and 370-480. In someembodiments, the polynucleic acid molecule consists of a target sequenceas set forth in SEQ ID NOs: 28-141 and 370-480.

In some embodiments, the polynucleic acid molecule comprises a sequencehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to a target sequence as setforth in SEQ ID NOs: 703-3406. In some embodiments, the polynucleic acidmolecule comprises a sequence having at least 50% sequence identity to atarget sequence as set forth in SEQ ID NOs: 703-3406. In someembodiments, the polynucleic acid molecule comprises a sequence havingat least 60% sequence identity to a target sequence as set forth in SEQID NOs: 703-3406. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 70% sequence identity to a targetsequence as set forth in SEQ ID NOs: 703-3406. In some embodiments, thepolynucleic acid molecule comprises a sequence having at least 75%sequence identity to a target sequence as set forth in SEQ ID NOs:703-3406. In some embodiments, the polynucleic acid molecule comprises asequence having at least 80% sequence identity to a target sequence asset forth in SEQ ID NOs: 703-3406. In some embodiments, the polynucleicacid molecule comprises a sequence having at least 85% sequence identityto a target sequence as set forth in SEQ ID NOs: 703-3406. In someembodiments, the polynucleic acid molecule comprises a sequence havingat least 90% sequence identity to a target sequence as set forth in SEQID NOs: 703-3406. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 95% sequence identity to a targetsequence as set forth in SEQ ID NOs: 703-3406. In some embodiments, thepolynucleic acid molecule comprises a sequence having at least 96%sequence identity to a target sequence as set forth in SEQ ID NOs:703-3406. In some embodiments, the polynucleic acid molecule comprises asequence having at least 97% sequence identity to a target sequence asset forth in SEQ ID NOs: 703-3406. In some embodiments, the polynucleicacid molecule comprises a sequence having at least 98% sequence identityto a target sequence as set forth in SEQ ID NOs: 703-3406. In someembodiments, the polynucleic acid molecule comprises a sequence havingat least 99% sequence identity to a target sequence as set forth in SEQID NOs: 703-3406. In some embodiments, the polynucleic acid moleculeconsists of a target sequence as set forth in SEQ ID NOs: 703-3406.

In some embodiments, the polynucleic acid molecule comprises a firstpolynucleotide and a second polynucleotide. In some instances, the firstpolynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a target sequence as set forth in SEQ ID NOs: 142-255,256-369, 481-591, 592-702, and 3407-14222. In some cases, the secondpolynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a target sequence as set forth in SEQ ID NOs: 142-255,256-369, 481-591, 592-702, and 3407-14222. In some cases, thepolynucleic acid molecule comprises a first polynucleotide having atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to a target sequence as set forth in SEQID NOs: 142-255, 481-591, 3407-6110, and 8815-11518, and a secondpolynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a targetsequence as set forth in SEQ ID NOs: 256-369, 592-702, 6111-8814, and11519-14222.

In some embodiments, the polynucleic acid molecule comprises a sensestrand (e.g., a passenger strand) and an antisense strand (e.g., a guidestrand). In some instances, the sense strand (e.g., the passengerstrand) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto a target sequence as set forth in SEQ ID NOs: 142-255, 481-591,3407-6110, and 8815-11518. In some instances, the antisense strand(e.g., the guide strand) comprises a sequence having at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to a target sequence as set forth in SEQ ID NOs:256-369, 592-702, 6111-8814, and 11519-14222.

In some embodiments, the polynucleic acid molecule described hereincomprises RNA or DNA. In some cases, the polynucleic acid moleculecomprises RNA. In some instances, RNA comprises short interfering RNA(siRNA), short hairpin RNA (shRNA), microRNA (miRNA), double-strandedRNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heterogeneousnuclear RNA (hnRNA). In some instances, RNA comprises shRNA. In someinstances, RNA comprises miRNA. In some instances, RNA comprises dsRNA.In some instances, RNA comprises tRNA. In some instances, RNA comprisesrRNA. In some instances, RNA comprises hnRNA. In some instances, the RNAcomprises siRNA. In some instances, the polynucleic acid moleculecomprises siRNA.

In some embodiments, the polynucleic acid molecule is from about 10 toabout 50 nucleotides in length. In some instances, the polynucleic acidmolecule is from about 10 to about 30, from about 15 to about 30, fromabout 18 to about 25, form about 18 to about 24, from about 19 to about23, or from about 20 to about 22 nucleotides in length.

In some embodiments, the polynucleic acid molecule is about 50nucleotides in length. In some instances, the polynucleic acid moleculeis about 45 nucleotides in length. In some instances, the polynucleicacid molecule is about 40 nucleotides in length. In some instances, thepolynucleic acid molecule is about 35 nucleotides in length. In someinstances, the polynucleic acid molecule is about 30 nucleotides inlength. In some instances, the polynucleic acid molecule is about 25nucleotides in length. In some instances, the polynucleic acid moleculeis about 20 nucleotides in length. In some instances, the polynucleicacid molecule is about 19 nucleotides in length. In some instances, thepolynucleic acid molecule is about 18 nucleotides in length. In someinstances, the polynucleic acid molecule is about 17 nucleotides inlength. In some instances, the polynucleic acid molecule is about 16nucleotides in length. In some instances, the polynucleic acid moleculeis about 15 nucleotides in length. In some instances, the polynucleicacid molecule is about 14 nucleotides in length. In some instances, thepolynucleic acid molecule is about 13 nucleotides in length. In someinstances, the polynucleic acid molecule is about 12 nucleotides inlength. In some instances, the polynucleic acid molecule is about 11nucleotides in length. In some instances, the polynucleic acid moleculeis about 10 nucleotides in length. In some instances, the polynucleicacid molecule is between about 10 and about 50 nucleotides in length. Insome instances, the polynucleic acid molecule is between about 10 andabout 45 nucleotides in length. In some instances, the polynucleic acidmolecule is between about 10 and about 40 nucleotides in length. In someinstances, the polynucleic acid molecule is between about 10 and about35 nucleotides in length. In some instances, the polynucleic acidmolecule is between about 10 and about 30 nucleotides in length. In someinstances, the polynucleic acid molecule is between about 10 and about25 nucleotides in length. In some instances, the polynucleic acidmolecule is between about 10 and about 20 nucleotides in length. In someinstances, the polynucleic acid molecule is between about 15 and about25 nucleotides in length. In some instances, the polynucleic acidmolecule is between about 15 and about 30 nucleotides in length. In someinstances, the polynucleic acid molecule is between about 12 and about30 nucleotides in length.

In some embodiments, the polynucleic acid molecule comprises a firstpolynucleotide. In some instances, the polynucleic acid moleculecomprises a second polynucleotide. In some instances, the polynucleicacid molecule comprises a first polynucleotide and a secondpolynucleotide. In some instances, the first polynucleotide is a sensestrand or passenger strand. In some instances, the second polynucleotideis an antisense strand or guide strand.

In some embodiments, the polynucleic acid molecule is a firstpolynucleotide. In some embodiments, the first polynucleotide is fromabout 10 to about 50 nucleotides in length. In some instances, the firstpolynucleotide is from about 10 to about 30, from about 15 to about 30,from about 18 to about 25, form about 18 to about 24, from about 19 toabout 23, or from about 20 to about 22 nucleotides in length.

In some instances, the first polynucleotide is about 50 nucleotides inlength. In some instances, the first polynucleotide is about 45nucleotides in length. In some instances, the first polynucleotide isabout 40 nucleotides in length. In some instances, the firstpolynucleotide is about 35 nucleotides in length. In some instances, thefirst polynucleotide is about 30 nucleotides in length. In someinstances, the first polynucleotide is about 25 nucleotides in length.In some instances, the first polynucleotide is about 20 nucleotides inlength. In some instances, the first polynucleotide is about 19nucleotides in length. In some instances, the first polynucleotide isabout 18 nucleotides in length. In some instances, the firstpolynucleotide is about 17 nucleotides in length. In some instances, thefirst polynucleotide is about 16 nucleotides in length. In someinstances, the first polynucleotide is about 15 nucleotides in length.In some instances, the first polynucleotide is about 14 nucleotides inlength. In some instances, the first polynucleotide is about 13nucleotides in length. In some instances, the first polynucleotide isabout 12 nucleotides in length. In some instances, the firstpolynucleotide is about 11 nucleotides in length. In some instances, thefirst polynucleotide is about 10 nucleotides in length. In someinstances, the first polynucleotide is between about 10 and about 50nucleotides in length. In some instances, the first polynucleotide isbetween about 10 and about 45 nucleotides in length. In some instances,the first polynucleotide is between about 10 and about 40 nucleotides inlength. In some instances, the first polynucleotide is between about 10and about 35 nucleotides in length. In some instances, the firstpolynucleotide is between about 10 and about 30 nucleotides in length.In some instances, the first polynucleotide is between about 10 andabout 25 nucleotides in length. In some instances, the firstpolynucleotide is between about 10 and about 20 nucleotides in length.In some instances, the first polynucleotide is between about 15 andabout 25 nucleotides in length. In some instances, the firstpolynucleotide is between about 15 and about 30 nucleotides in length.In some instances, the first polynucleotide is between about 12 andabout 30 nucleotides in length.

In some embodiments, the polynucleic acid molecule is a secondpolynucleotide. In some embodiments, the second polynucleotide is fromabout 10 to about 50 nucleotides in length. In some instances, thesecond polynucleotide is from about 10 to about 30, from about 15 toabout 30, from about 18 to about 25, form about 18 to about 24, fromabout 19 to about 23, or from about 20 to about 22 nucleotides inlength.

In some instances, the second polynucleotide is about 50 nucleotides inlength. In some instances, the second polynucleotide is about 45nucleotides in length. In some instances, the second polynucleotide isabout 40 nucleotides in length. In some instances, the secondpolynucleotide is about 35 nucleotides in length. In some instances, thesecond polynucleotide is about 30 nucleotides in length. In someinstances, the second polynucleotide is about 25 nucleotides in length.In some instances, the second polynucleotide is about 20 nucleotides inlength. In some instances, the second polynucleotide is about 19nucleotides in length. In some instances, the second polynucleotide isabout 18 nucleotides in length. In some instances, the secondpolynucleotide is about 17 nucleotides in length. In some instances, thesecond polynucleotide is about 16 nucleotides in length. In someinstances, the second polynucleotide is about 15 nucleotides in length.In some instances, the second polynucleotide is about 14 nucleotides inlength. In some instances, the second polynucleotide is about 13nucleotides in length. In some instances, the second polynucleotide isabout 12 nucleotides in length. In some instances, the secondpolynucleotide is about 11 nucleotides in length. In some instances, thesecond polynucleotide is about 10 nucleotides in length. In someinstances, the second polynucleotide is between about 10 and about 50nucleotides in length. In some instances, the second polynucleotide isbetween about 10 and about 45 nucleotides in length. In some instances,the second polynucleotide is between about 10 and about 40 nucleotidesin length. In some instances, the second polynucleotide is between about10 and about 35 nucleotides in length. In some instances, the secondpolynucleotide is between about 10 and about 30 nucleotides in length.In some instances, the second polynucleotide is between about 10 andabout 25 nucleotides in length. In some instances, the secondpolynucleotide is between about 10 and about 20 nucleotides in length.In some instances, the second polynucleotide is between about 15 andabout 25 nucleotides in length. In some instances, the secondpolynucleotide is between about 15 and about 30 nucleotides in length.In some instances, the second polynucleotide is between about 12 andabout 30 nucleotides in length.

In some embodiments, the polynucleic acid molecule comprises a firstpolynucleotide and a second polynucleotide. In some instances, thepolynucleic acid molecule further comprises a blunt terminus, anoverhang, or a combination thereof. In some instances, the bluntterminus is a 5′ blunt terminus, a 3′ blunt terminus, or both. In somecases, the overhang is a 5′ overhang, 3′ overhang, or both. In somecases, the overhang comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-basepairing nucleotides. In some cases, the overhang comprises 1, 2, 3, 4,5, or 6 non-base pairing nucleotides. In some cases, the overhangcomprises 1, 2, 3, or 4 non-base pairing nucleotides. In some cases, theoverhang comprises 1 non-base pairing nucleotide. In some cases, theoverhang comprises 2 non-base pairing nucleotides. In some cases, theoverhang comprises 3 non-base pairing nucleotides. In some cases, theoverhang comprises 4 non-base pairing nucleotides.

In some embodiments, the sequence of the polynucleic acid molecule is atleast 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%,or 99.5% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least50% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least60% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least70% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least80% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least90% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least95% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least99% complementary to a target sequence described herein. In someinstances, the sequence of the polynucleic acid molecule is 100%complementary to a target sequence described herein.

In some embodiments, the sequence of the polynucleic acid molecule has 5or less mismatches to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule has 4 or lessmismatches to a target sequence described herein. In some instances, thesequence of the polynucleic acid molecule has 3 or less mismatches to atarget sequence described herein. In some cases, the sequence of thepolynucleic acid molecule has 2 or less mismatches to a target sequencedescribed herein. In some cases, the sequence of the polynucleic acidmolecule has 1 or less mismatches to a target sequence described herein.

In some embodiments, the specificity of the polynucleic acid moleculethat hybridizes to a target sequence described herein is a 95%, 98%,99%, 99.5% or 100% sequence complementarity of the polynucleic acidmolecule to a target sequence. In some instances, the hybridization is ahigh stringent hybridization condition.

In some embodiments, the polynucleic acid molecule has reducedoff-target effect. In some instances, “off-target” or “off-targeteffects” refer to any instance in which a polynucleic acid polymerdirected against a given target causes an unintended effect byinteracting either directly or indirectly with another mRNA sequence, aDNA sequence or a cellular protein or other moiety. In some instances,an “off-target effect” occurs when there is a simultaneous degradationof other transcripts due to partial homology or complementarity betweenthat other transcript and the sense and/or antisense strand of thepolynucleic acid molecule.

In some embodiments, the polynucleic acid molecule comprises natural orsynthetic or artificial nucleotide analogues or bases. In some cases,the polynucleic acid molecule comprises combinations of DNA, RNA and/ornucleotide analogues. In some instances, the synthetic or artificialnucleotide analogues or bases comprise modifications at one or more ofribose moiety, phosphate moiety, nucleoside moiety, or a combinationthereof.

In some embodiments, nucleotide analogues or artificial nucleotide basecomprise a nucleic acid with a modification at a 2′ hydroxyl group ofthe ribose moiety. In some instances, the modification includes an H,OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety.Exemplary alkyl moiety includes, but is not limited to, halogens,sulfurs, thiols, thioethers, thioesters, amines (primary, secondary, ortertiary), amides, ethers, esters, alcohols and oxygen. In someinstances, the alkyl moiety further comprises a modification. In someinstances, the modification comprises an azo group, a keto group, analdehyde group, a carboxyl group, a nitro group, a nitroso, group, anitrile group, a heterocycle (e.g., imidazole, hydrazino orhydroxylamino) group, an isocyanate or cyanate group, or a sulfurcontaining group (e.g., sulfoxide, sulfone, sulfide, and disulfide). Insome instances, the alkyl moiety further comprises a heterosubstitution. In some instances, the carbon of the heterocyclic group issubstituted by a nitrogen, oxygen or sulfur. In some instances, theheterocyclic substitution includes but is not limited to, morpholino,imidazole, and pyrrolidino.

In some instances, the modification at the 2′ hydroxyl group is a2′-O-methyl modification or a 2′-O-methoxyethyl (2′-O-MOE) modification.In some cases, the 2′-O-methyl modification adds a methyl group to the2′ hydroxyl group of the ribose moiety whereas the 2′O-methoxyethylmodification adds a methoxyethyl group to the 2′ hydroxyl group of theribose moiety. Exemplary chemical structures of a 2′-O-methylmodification of an adenosine molecule and 2′O-methoxyethyl modificationof an uridine are illustrated below.

In some instances, the modification at the 2′ hydroxyl group is a2′-O-aminopropyl modification in which an extended amine groupcomprising a propyl linker binds the amine group to the 2′ oxygen. Insome instances, this modification neutralizes the phosphate derivedoverall negative charge of the oligonucleotide molecule by introducingone positive charge from the amine group per sugar and thereby improvescellular uptake properties due to its zwitterionic properties. Anexemplary chemical structure of a 2′-O-aminopropyl nucleosidephosphoramidite is illustrated below.

In some instances, the modification at the 2′ hydroxyl group is a lockedor bridged ribose modification (e.g., locked nucleic acid or LNA) inwhich the oxygen molecule bound at the 2′ carbon is linked to the 4′carbon by a methylene group, thus forming a2′-C,4′-C-oxy-methylene-linked bicyclic ribonucleotide monomer.Exemplary representations of the chemical structure of LNA areillustrated below. The representation shown to the left highlights thechemical connectivities of an LNA monomer. The representation shown tothe right highlights the locked 3′-endo (³E) conformation of thefuranose ring of an LNA monomer.

In some instances, the modification at the 2′ hydroxyl group comprisesethylene nucleic acids (ENA) such as for example 2′-4′-ethylene-bridgednucleic acid, which locks the sugar conformation into a C₃′-endo sugarpuckering conformation. ENA are part of the bridged nucleic acids classof modified nucleic acids that also comprises LNA. Exemplary chemicalstructures of the ENA and bridged nucleic acids are illustrated below.

In some embodiments, additional modifications at the 2′ hydroxyl groupinclude 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA).

In some embodiments, nucleotide analogues comprise modified bases suchas, but not limited to, 5-propynyluridine, 5-propynylcytidine,6-methyladenine, 6-methylguanine, N, N, -dimethyladenine,2-propyladenine, 2propylguanine, 2-aminoadenine, 1-methylinosine,3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotideshaving a modification at the 5 position, 5-(2-amino) propyl uridine,5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine,2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine,7-methylguanosine, 2, 2-dimethylguanosine, 5-methylaminoethyluridine,5-methyloxyuridine, deazanucleotides such as 7-deaza-adenosine,6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine,other thio bases such as 2-thiouridine and 4-thiouridine and2-thiocytidine, dihydrouridine, pseudouridine, queuosine, archaeosine,naphthyl and substituted naphthyl groups, any O- and N-alkylated purinesand pyrimidines such as N6-methyladenosine,5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one,pyridine-2-one, phenyl and modified phenyl groups such as aminophenol or2,4, 6-trimethoxy benzene, modified cytosines that act as G-clampnucleotides, 8-substituted adenines and guanines, 5-substituted uracilsand thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides,carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylatednucleotides. Modified nucleotides also include those nucleotides thatare modified with respect to the sugar moiety, as well as nucleotideshaving sugars or analogs thereof that are not ribosyl. For example, thesugar moieties, in some cases are or be based on, mannoses, arabinoses,glucopyranoses, galactopyranoses, 4′-thioribose, and other sugars,heterocycles, or carbocycles. The term nucleotide also includes what areknown in the art as universal bases. By way of example, universal basesinclude but are not limited to 3-nitropyrrole, 5-nitroindole, ornebularine.

In some embodiments, nucleotide analogues further comprise morpholinos,peptide nucleic acids (PNAs), methylphosphonate nucleotides,thiolphosphonate nucleotides, 2′-fluoro N3-P5′-phosphoramidites, 1′,5′-anhydrohexitol nucleic acids (HNAs), or a combination thereof.Morpholino or phosphorodiamidate morpholino oligo (PMO) comprisessynthetic molecules whose structure mimics natural nucleic acidstructure by deviates from the normal sugar and phosphate structures. Insome instances, the five member ribose ring is substituted with a sixmember morpholino ring containing four carbons, one nitrogen and oneoxygen. In some cases, the ribose monomers are linked by aphosphordiamidate group instead of a phosphate group. In such cases, thebackbone alterations remove all positive and negative charges makingmorpholinos neutral molecules capable of crossing cellular membraneswithout the aid of cellular delivery agents such as those used bycharged oligonucleotides.

In some embodiments, peptide nucleic acid (PNA) does not contain sugarring or phosphate linkage and the bases are attached and appropriatelyspaced by oligoglycine-like molecules, therefore, eliminating a backbonecharge.

In some embodiments, one or more modifications optionally occur at theinternucleotide linkage. In some instances, modified internucleotidelinkage include, but is not limited to, phosphorothioates,phosphorodithioates, methylphosphonates, 5′-alkylenephosphonates,5′-methylphosphonate, 3′-alkylene phosphonates, borontrifluoridates,borano phosphate esters and selenophosphates of 3′-5′linkage or2′-5′linkage, phosphotriesters, thionoalkylphosphotriesters, hydrogenphosphonate linkages, alkyl phosphonates, alkylphosphonothioates,arylphosphonothioates, phosphoroselenoates, phosphorodiselenoates,phosphinates, phosphoramidates, 3′-alkylphosphoramidates,aminoalkylphosphoramidates, thionophosphoramidates,phosphoropiperazidates, phosphoroanilothioates, phosphoroanilidates,ketones, sulfones, sulfonamides, carbonates, carbamates,methylenehydrazos, methylenedimethylhydrazos, formacetals,thioformacetals, oximes, methyleneiminos, methylenemethyliminos,thioamidates, linkages with riboacetyl groups, aminoethyl glycine, silylor siloxane linkages, alkyl or cycloalkyl linkages with or withoutheteroatoms of, for example, 1 to 10 carbons that are saturated orunsaturated and/or substituted and/or contain heteroatoms, linkages withmorpholino structures, amides, polyamides wherein the bases are attachedto the aza nitrogens of the backbone directly or indirectly, andcombinations thereof. Phosphorothioate antisene oligonucleotides (PSASO) are antisense oligonucleotides comprising a phosphorothioatelinkage. An exemplary PS ASO is illustrated below.

In some instances, the modification is a methyl or thiol modificationsuch as methylphosphonate or thiolphosphonate modification. Exemplarythiolphosphonate nucleotide (left) and methylphosphonate nucleotide(right) are illustrated below.

In some instances, a modified nucleotide includes, but is not limitedto, 2′-fluoro N3-P5′-phosphoramidites illustrated as:

In some instances, a modified nucleotide includes, but is not limitedto, hexitol nucleic acid (or 1′, 5′-anhydrohexitol nucleic acids (HNA))illustrated as:

In some embodiments, one or more modifications further optionallyinclude modifications of the ribose moiety, phosphate backbone and thenucleoside, or modifications of the nucleotide analogues at the 3′ orthe 5′ terminus. For example, the 3′ terminus optionally include a 3′cationic group, or by inverting the nucleoside at the 3′-terminus with a3-3′ linkage. In another alternative, the 3′-terminus is optionallyconjugated with an aminoalkyl group, e.g., a 3′ C5-aminoalkyl dT. In anadditional alternative, the 3′-terminus is optionally conjugated with anabasic site, e.g., with an apurinic or apyrimidinic site. In someinstances, the 5-terminus is conjugated with an aminoalkyl group, e.g.,a 5-O-alkylamino substituent. In some cases, the 5′-terminus isconjugated with an abasic site, e.g., with an apurinic or apyrimidinicsite.

In some embodiments, the polynucleic acid molecule comprises one or moreof the artificial nucleotide analogues described herein. In someinstances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of theartificial nucleotide analogues described herein. In some embodiments,the artificial nucleotide analogues include 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, 2′-fluoroN3-P5′-phosphoramidites, or a combination thereof. In some instances,the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of the artificialnucleotide analogues selected from 2′-O-methyl, 2′-O-methoxyethyl(2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro,2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified, LNA, ENA,PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonatenucleotides, 2′-fluoro N3-P5′-phosphoramidites, or a combinationthereof. In some instances, the polynucleic acid molecule comprises 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, ormore of 2′-O-methyl modified nucleotides. In some instances, thepolynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 20, 25, or more of 2′-O-methoxyethyl(2′-O-MOE) modified nucleotides. In some instances, the polynucleic acidmolecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 20, 25, or more of thiolphosphonate nucleotides.

In some instances, the polynucleic acid molecule comprises at least oneof: from about 5% to about 100% modification, from about 10% to about100% modification, from about 20% to about 100% modification, from about30% to about 100% modification, from about 40% to about 100%modification, from about 50% to about 100% modification, from about 60%to about 100% modification, from about 70% to about 100% modification,from about 80% to about 100% modification, and from about 90% to about100% modification.

In some cases, the polynucleic acid molecule comprises at least one of:from about 10% to about 90% modification, from about 20% to about 90%modification, from about 30% to about 90% modification, from about 40%to about 90% modification, from about 50% to about 90% modification,from about 60% to about 90% modification, from about 70% to about 90%modification, and from about 80% to about 100% modification.

In some cases, the polynucleic acid molecule comprises at least one of:from about 10% to about 80% modification, from about 20% to about 80%modification, from about 30% to about 80% modification, from about 40%to about 80% modification, from about 50% to about 80% modification,from about 60% to about 80% modification, and from about 70% to about80% modification.

In some instances, the polynucleic acid molecule comprises at least oneof: from about 10% to about 70% modification, from about 20% to about70% modification, from about 30% to about 70% modification, from about40% to about 70% modification, from about 50% to about 70% modification,and from about 60% to about 70% modification.

In some instances, the polynucleic acid molecule comprises at least oneof: from about 10% to about 60% modification, from about 20% to about60% modification, from about 30% to about 60% modification, from about40% to about 60% modification, and from about 50% to about 60%modification.

In some cases, the polynucleic acid molecule comprises at least one of:from about 10% to about 50% modification, from about 20% to about 50%modification, from about 30% to about 50% modification, and from about40% to about 50% modification.

In some cases, the polynucleic acid molecule comprises at least one of:from about 10% to about 40% modification, from about 20% to about 40%modification, and from about 30% to about 40% modification.

In some cases, the polynucleic acid molecule comprises at least one of:from about 10% to about 30% modification, and from about 20% to about30% modification.

In some cases, the polynucleic acid molecule comprises from about 10% toabout 20% modification.

In some cases, the polynucleic acid molecule comprises from about 15% toabout 90%, from about 20% to about 80%, from about 30% to about 70%, orfrom about 40% to about 60% modifications.

In additional cases, the polynucleic acid molecule comprises at leastabout 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%modification.

In some embodiments, the polynucleic acid molecule comprises at leastabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22 ormore modifications.

In some instances, the polynucleic acid molecule comprises at leastabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22 ormore modified nucleotides.

In some instances, from about 5 to about 100% of the polynucleic acidmolecule comprise the artificial nucleotide analogues described herein.In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of thepolynucleic acid molecule comprise the artificial nucleotide analoguesdescribed herein. In some instances, about 5% of the polynucleic acidmolecule comprises the artificial nucleotide analogues described herein.In some instances, about 10% of the polynucleic acid molecule comprisesthe artificial nucleotide analogues described herein. In some instances,about 15% of the polynucleic acid molecule comprises the artificialnucleotide analogues described herein. In some instances, about 20% ofthe polynucleic acid molecule comprises the artificial nucleotideanalogues described herein. In some instances, about 25% of thepolynucleic acid molecule comprises the artificial nucleotide analoguesdescribed herein. In some instances, about 30% of the polynucleic acidmolecule comprises the artificial nucleotide analogues described herein.In some instances, about 35% of the polynucleic acid molecule comprisesthe artificial nucleotide analogues described herein. In some instances,about 40% of the polynucleic acid molecule comprises the artificialnucleotide analogues described herein. In some instances, about 45% ofthe polynucleic acid molecule comprises the artificial nucleotideanalogues described herein. In some instances, about 50% of thepolynucleic acid molecule comprises the artificial nucleotide analoguesdescribed herein. In some instances, about 55% of the polynucleic acidmolecule comprises the artificial nucleotide analogues described herein.In some instances, about 60% of the polynucleic acid molecule comprisesthe artificial nucleotide analogues described herein. In some instances,about 65% of the polynucleic acid molecule comprises the artificialnucleotide analogues described herein. In some instances, about 70% ofthe polynucleic acid molecule comprises the artificial nucleotideanalogues described herein. In some instances, about 75% of thepolynucleic acid molecule comprises the artificial nucleotide analoguesdescribed herein. In some instances, about 80% of the polynucleic acidmolecule comprises the artificial nucleotide analogues described herein.In some instances, about 85% of the polynucleic acid molecule comprisesthe artificial nucleotide analogues described herein. In some instances,about 90% of the polynucleic acid molecule comprises the artificialnucleotide analogues described herein. In some instances, about 95% ofthe polynucleic acid molecule comprises the artificial nucleotideanalogues described herein. In some instances, about 96% of thepolynucleic acid molecule comprises the artificial nucleotide analoguesdescribed herein. In some instances, about 97% of the polynucleic acidmolecule comprises the artificial nucleotide analogues described herein.In some instances, about 98% of the polynucleic acid molecule comprisesthe artificial nucleotide analogues described herein. In some instances,about 99% of the polynucleic acid molecule comprises the artificialnucleotide analogues described herein. In some instances, about 100% ofthe polynucleic acid molecule comprises the artificial nucleotideanalogues described herein. In some embodiments, the artificialnucleotide analogues include 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl(2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified, LNA, ENA,PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonatenucleotides, 2′-fluoro N3-P5′-phosphoramidites, or a combinationthereof.

In some embodiments, the polynucleic acid molecule comprises from about1 to about 25 modifications in which the modification comprises anartificial nucleotide analogues described herein. In some embodiments,the polynucleic acid molecule comprises about 1 modification in whichthe modification comprises an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 2 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 3 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 4 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 5 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 6 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 7 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 8 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 9 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 10 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 11 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 12 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 13 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 14 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 15 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 16 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 17 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 18 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 19 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 20 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 21 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 22 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 23 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 24 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 25 modifications in which themodifications comprise an artificial nucleotide analogue describedherein.

In some embodiments, a polynucleic acid molecule is assembled from twoseparate polynucleotides wherein one polynucleotide comprises the sensestrand and the second polynucleotide comprises the antisense strand ofthe polynucleic acid molecule. In other embodiments, the sense strand isconnected to the antisense strand via a linker molecule, which in someinstances is a polynucleotide linker or a non-nucleotide linker.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and antisense strand, wherein pyrimidine nucleotides in the sensestrand comprises 2′-O-methylpyrimidine nucleotides and purinenucleotides in the sense strand comprise 2′-deoxy purine nucleotides. Insome embodiments, a polynucleic acid molecule comprises a sense strandand antisense strand, wherein pyrimidine nucleotides present in thesense strand comprise 2′-deoxy-2′-fluoro pyrimidine nucleotides andwherein purine nucleotides present in the sense strand comprise 2′-deoxypurine nucleotides.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and antisense strand, wherein the pyrimidine nucleotides whenpresent in said antisense strand are 2′-deoxy-2′-fluoro pyrimidinenucleotides and the purine nucleotides when present in said antisensestrand are 2′-O-methyl purine nucleotides.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and antisense strand, wherein the pyrimidine nucleotides whenpresent in said antisense strand are 2′-deoxy-2′-fluoro pyrimidinenucleotides and wherein the purine nucleotides when present in saidantisense strand comprise 2′-deoxy-purine nucleotides.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and antisense strand, wherein the sense strand includes aterminal cap moiety at the 5′-end, the 3′-end, or both of the 5′ and 3′ends of the sense strand. In other embodiments, the terminal cap moietyis an inverted deoxy abasic moiety.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and an antisense strand, wherein the antisense strand comprises aphosphate backbone modification at the 3′ end of the antisense strand.In some instances, the phosphate backbone modification is aphosphorothioate.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and an antisense strand, wherein the antisense strand comprises aglyceryl modification at the 3′ end of the antisense strand.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and an antisense strand, in which the sense strand comprises oneor more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or more phosphorothioate internucleotidelinkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and in which the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the antisense strand. In otherembodiments, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more, pyrimidine nucleotides of the sense and/or antisense strandare chemically-modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′- and 5′-ends, being present in the same ordifferent strand.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and an antisense strand, in which the sense strand comprisesabout 1 to about 25, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioateinternucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of thesense strand; and in which the antisense strand comprises about 1 toabout 25 or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioateinternucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe antisense strand. In other embodiments, one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides ofthe sense and/or antisense strand are chemically-modified with 2′-deoxy,2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without about1 to about 25 or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′- and 5′-ends, being present in the same ordifferent strand.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and an antisense strand, in which the antisense strand comprisesone or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate internucleotidelinkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In other embodiments, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, or more pyrimidine nucleotides of the sense and/or antisensestrand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without one or more, forexample, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends, being present in the same ordifferent strand.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and an antisense strand, in which the antisense strand comprisesabout 1 to about 25 or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioateinternucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe sense strand; and wherein the antisense strand comprises about 1 toabout 25 or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioateinternucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe antisense strand. In other embodiments, one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides ofthe sense and/or antisense strand are chemically-modified with 2′-deoxy,2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without about1 to about 5, for example about 1, 2, 3, 4, 5 or more phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′- and 5′-ends, being present in the same ordifferent strand.

In some embodiments, a polynucleic acid molecule described herein is achemically-modified short interfering nucleic acid molecule having about1 to about 25, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate internucleotidelinkages in each strand of the polynucleic acid molecule.

In another embodiment, a polynucleic acid molecule described hereincomprises 2′-5′ internucleotide linkages. In some instances, the 2′-5′internucleotide linkage(s) is at the 3′-end, the 5′-end, or both of the3′- and 5′-ends of one or both sequence strands. In addition instances,the 2′-5′ internucleotide linkage(s) is present at various otherpositions within one or both sequence strands, for example, about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkageof a pyrimidine nucleotide in one or both strands of the polynucleicacid molecule comprise a 2′-5′ internucleotide linkage, or about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkageof a purine nucleotide in one or both strands of the polynucleic acidmolecule comprise a 2′-5′ internucleotide linkage.

In some embodiments, a polynucleic acid molecule is a single strandedpolynucleic acid molecule that mediates RNAi activity in a cell orreconstituted in vitro system, wherein the polynucleic acid moleculecomprises a single stranded polynucleotide having complementarity to atarget nucleic acid sequence, and wherein one or more pyrimidinenucleotides present in the polynucleic acid are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any purine nucleotides present in the polynucleic acid are2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are2′-deoxy purine nucleotides or alternately a plurality of purinenucleotides are 2′-deoxy purine nucleotides), and a terminal capmodification, that is optionally present at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the antisense sequence, the polynucleicacid molecule optionally further comprising about 1 to about 4 (e.g.,about 1, 2, 3, or 4) terminal 2′-deoxynucleotides at the 3′-end of thepolynucleic acid molecule, wherein the terminal nucleotides furthercomprise one or more (e.g., 1, 2, 3, or 4) phosphorothioateinternucleotide linkages, and wherein the polynucleic acid moleculeoptionally further comprises a terminal phosphate group, such as a5′-terminal phosphate group.

In some cases, one or more of the artificial nucleotide analoguesdescribed herein are resistant toward nucleases such as for exampleribonuclease such as RNase H, deoxyribonuclease such as DNase, orexonuclease such as 5′-3′ exonuclease and 3′-5′ exonuclease whencompared to natural polynucleic acid molecules. In some instances,artificial nucleotide analogues comprising 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, 2′-fluoroN3-P5′-phosphoramidites, or combinations thereof are resistant towardnucleases such as for example ribonuclease such as RNase H,deoxyribonuclease such as DNase, or exonuclease such as 5′-3′exonuclease and 3′-5′ exonuclease. In some instances, 2′-O-methylmodified polynucleic acid molecule is nuclease resistance (e.g., RNaseH, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance). In someinstances, 2′O-methoxyethyl (2′-O-MOE) modified polynucleic acidmolecule is nuclease resistance (e.g., RNase H, DNase, 5′-3′ exonucleaseor 3′-5′ exonuclease resistance). In some instances, 2′-O-aminopropylmodified polynucleic acid molecule is nuclease resistance (e.g., RNaseH, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance). In someinstances, 2′-deoxy modified polynucleic acid molecule is nucleaseresistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). In some instances, T-deoxy-2′-fluoro modified polynucleicacid molecule is nuclease resistance (e.g., RNase H, DNase, 5′-3′exonuclease or 3′-5′ exonuclease resistance). In some instances,2′-O-aminopropyl (2′-O-AP) modified polynucleic acid molecule isnuclease resistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistance). In some instances, 2′-O-dimethylaminoethyl(2′-O-DMAOE) modified polynucleic acid molecule is nuclease resistance(e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). In some instances, 2′-O-dimethylaminopropyl (2′-O-DMAP)modified polynucleic acid molecule is nuclease resistance (e.g., RNaseH, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance). In someinstances, T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE) modifiedpolynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase,5′-3′ exonuclease or 3′-5′ exonuclease resistance). In some instances,2′-O—N-methylacetamido (2′-O-NMA) modified polynucleic acid molecule isnuclease resistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistance). In some instances, LNA modified polynucleicacid molecule is nuclease resistance (e.g., RNase H, DNase, 5′-3′exonuclease or 3′-5′ exonuclease resistance). In some instances, ENAmodified polynucleic acid molecule is nuclease resistance (e.g., RNaseH, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance). In someinstances, HNA modified polynucleic acid molecule is nuclease resistance(e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). In some instances, morpholinos is nuclease resistance(e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). In some instances, PNA modified polynucleic acid moleculeis resistant to nucleases (e.g., RNase H, DNase, 5′-3′ exonuclease or3′-5′ exonuclease resistance). In some instances, methylphosphonatenucleotides modified polynucleic acid molecule is nuclease resistance(e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). In some instances, thiolphosphonate nucleotides modifiedpolynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase,5′-3′ exonuclease or 3′-5′ exonuclease resistance). In some instances,polynucleic acid molecule comprising 2′-fluoro N3-P5′-phosphoramiditesis nuclease resistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistance). In some instances, the 5′ conjugates describedherein inhibit 5′-3′ exonucleolytic cleavage. In some instances, the 3′conjugates described herein inhibit 3-5′ exonucleolytic cleavage.

In some embodiments, one or more of the artificial nucleotide analoguesdescribed herein have increased binding affinity toward their mRNAtarget relative to an equivalent natural polynucleic acid molecule. Theone or more of the artificial nucleotide analogues comprising2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, or 2′-fluoroN3-P5′-phosphoramidites have increased binding affinity toward theirmRNA target relative to an equivalent natural polynucleic acid molecule.In some instances, 2′-O-methyl modified polynucleic acid molecule hasincreased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-methoxyethyl (2′-O-MOE) modified polynucleic acid molecule hasincreased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-aminopropyl modified polynucleic acid molecule has increasedbinding affinity toward their mRNA target relative to an equivalentnatural polynucleic acid molecule. In some instances, 2′-deoxy modifiedpolynucleic acid molecule has increased binding affinity toward theirmRNA target relative to an equivalent natural polynucleic acid molecule.In some instances, T-deoxy-2′-fluoro modified polynucleic acid moleculehas increased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-aminopropyl (2′-O-AP) modified polynucleic acid molecule hasincreased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-dimethylaminoethyl (2′-O-DMAOE) modified polynucleic acid moleculehas increased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-dimethylaminopropyl (2′-O-DMAP) modified polynucleic acid moleculehas increased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE) modified polynucleic acidmolecule has increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid molecule. In someinstances, 2′-O—N-methylacetamido (2′-O-NMA) modified polynucleic acidmolecule has increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid molecule. In someinstances, LNA modified polynucleic acid molecule has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some instances, ENA modified polynucleicacid molecule has increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid molecule. In someinstances, PNA modified polynucleic acid molecule has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some instances, HNA modified polynucleicacid molecule has increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid molecule. In someinstances, morpholino modified polynucleic acid molecule has increasedbinding affinity toward their mRNA target relative to an equivalentnatural polynucleic acid molecule. In some instances, methylphosphonatenucleotides modified polynucleic acid molecule has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some instances, thiolphosphonatenucleotides modified polynucleic acid molecule has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some instances, polynucleic acid moleculecomprising 2′-fluoro N3-P5′-phosphoramidites has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some cases, the increased affinity isillustrated with a lower Kd, a higher melt temperature (Tm), or acombination thereof.

In some embodiments, a polynucleic acid molecule described herein is achirally pure (or stereo pure) polynucleic acid molecule, or apolynucleic acid molecule comprising a single enantiomer. In someinstances, the polynucleic acid molecule comprises L-nucleotide. In someinstances, the polynucleic acid molecule comprises D-nucleotides. Insome instance, a polynucleic acid molecule composition comprises lessthan 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its mirrorenantiomer. In some cases, a polynucleic acid molecule compositioncomprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or lessof a racemic mixture. In some instances, the polynucleic acid moleculeis a polynucleic acid molecule described in: U.S. Patent PublicationNos: 2014/194610 and 2015/211006; and PCT Publication No.: WO2015107425.

In some embodiments, a polynucleic acid molecule described herein isfurther modified to include an aptamer conjugating moiety. In someinstances, the aptamer conjugating moiety is a DNA aptamer conjugatingmoiety. In some instances, the aptamer conjugating moiety is Alphamer(Centauri Therapeutics), which comprises an aptamer portion thatrecognizes a specific cell-surface target and a portion that presents aspecific epitopes for attaching to circulating antibodies. In someinstance, a polynucleic acid molecule described herein is furthermodified to include an aptamer conjugating moiety as described in: U.S.Pat. Nos. 8,604,184, 8,591,910, and 7,850,975.

In additional embodiments, a polynucleic acid molecule described hereinis modified to increase its stability. In some embodiment, thepolynucleic acid molecule is RNA (e.g., siRNA). In some instances, thepolynucleic acid molecule is modified by one or more of themodifications described above to increase its stability. In some cases,the polynucleic acid molecule is modified at the 2′ hydroxyl position,such as by 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl,2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA) modification or by a locked or bridgedribose conformation (e.g., LNA or ENA). In some cases, the polynucleicacid molecule is modified by 2′-O-methyl and/or 2′-O-methoxyethylribose. In some cases, the polynucleic acid molecule also includesmorpholinos, PNAs, HNA, methylphosphonate nucleotides, thiolphosphonatenucleotides, and/or 2′-fluoro N3-P5′-phosphoramidites to increase itsstability. In some instances, the polynucleic acid molecule is achirally pure (or stereo pure) polynucleic acid molecule. In someinstances, the chirally pure (or stereo pure) polynucleic acid moleculeis modified to increase its stability. Suitable modifications to the RNAto increase stability for delivery will be apparent to the skilledperson.

In some instances, the polynucleic acid molecule is a double-strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. In some instances, the polynucleic acid molecule isassembled from two separate polynucleotides, where one strand is thesense strand and the other is the antisense strand, wherein theantisense and sense strands are self-complementary (e.g., each strandcomprises nucleotide sequence that is complementary to nucleotidesequence in the other strand; such as where the antisense strand andsense strand form a duplex or double stranded structure, for examplewherein the double stranded region is about 19, 20, 21, 22, 23, or morebase pairs); the antisense strand comprises nucleotide sequence that iscomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof and the sense strand comprises nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof.Alternatively, the polynucleic acid molecule is assembled from a singleoligonucleotide, where the self-complementary sense and antisenseregions of the polynucleic acid molecule are linked by means of anucleic acid based or non-nucleic acid-based linker(s).

In some cases, the polynucleic acid molecule is a polynucleotide with aduplex, asymmetric duplex, hairpin or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. In other cases, the polynucleic acid molecule is a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide is processed either in vivo or in vitro togenerate an active polynucleic acid molecule capable of mediating RNAi.In additional cases, the polynucleic acid molecule also comprises asingle-stranded polynucleotide having nucleotide sequence complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof (for example, where such polynucleic acid molecule does notrequire the presence within the polynucleic acid molecule of nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof), wherein the single stranded polynucleotide further comprises aterminal phosphate group, such as a 5′-phosphate (see for exampleMartinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002,Molecular Cell, 10, 537-568), or 5′,3′-diphosphate.

In some instances, an asymmetric hairpin is a linear polynucleic acidmolecule comprising an antisense region, a loop portion that comprisesnucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complimentary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin polynucleic acid molecule comprises an antisense region havinglength sufficient to mediate RNAi in a cell or in vitro system (e.g.about 19 to about 22 nucleotides) and a loop region comprising about 4to about 8 nucleotides, and a sense region having about 3 to about 18nucleotides that are complementary to the antisense region. In somecases, the asymmetric hairpin polynucleic acid molecule also comprises a5′-terminal phosphate group that is chemically modified. In additionalcases, the loop portion of the asymmetric hairpin polynucleic acidmolecule comprises nucleotides, non-nucleotides, linker molecules, orconjugate molecules.

In some embodiments, an asymmetric duplex is a polynucleic acid moleculehaving two separate strands comprising a sense region and an antisenseregion, wherein the sense region comprises fewer nucleotides than theantisense region to the extent that the sense region has enoughcomplimentary nucleotides to base pair with the antisense region andform a duplex. For example, an asymmetric duplex polynucleic acidmolecule comprises an antisense region having length sufficient tomediate RNAi in a cell or in vitro system (e.g. about 19 to about 22nucleotides) and a sense region having about 3 to about 18 nucleotidesthat are complementary to the antisense region.

In some cases, a universal base refers to nucleotide base analogs thatform base pairs with each of the natural DNA/RNA bases with littlediscrimination between them. Non-limiting examples of universal basesinclude C-phenyl, C-naphthyl and other aromatic derivatives, inosine,azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole,4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (seefor example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

Polynucleic Acid Molecule Synthesis

In some embodiments, a polynucleic acid molecule described herein isconstructed using chemical synthesis and/or enzymatic ligation reactionsusing procedures known in the art. For example, a polynucleic acidmolecule is chemically synthesized using naturally occurring nucleotidesor variously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the polynucleic acid molecule and target nucleicacids. Exemplary methods include those described in: U.S. Pat. Nos.5,142,047; 5,185,444; 5,889,136; 6,008,400; and 6,111,086; PCTPublication No. WO2009099942; or European Publication No. 1579015.Additional exemplary methods include those described in: Griffey et al.,“2′-O-aminopropyl ribonucleotides: a zwitterionic modification thatenhances the exonuclease resistance and biological activity of antisenseoligonucleotides,” J. Med. Chem. 39(26):5100-5109 (1997)); Obika, et al.“Synthesis of 2′-0,4′-C-methyleneuridine and -cytidine. Novel bicyclicnucleosides having a fixed C3, -endo sugar puckering”. TetrahedronLetters 38 (50): 8735 (1997); Koizumi, M. “ENA oligonucleotides astherapeutics”. Current opinion in molecular therapeutics 8 (2): 144-149(2006); and Abramova et al., “Novel oligonucleotide analogues based onmorpholino nucleoside subunits-antisense technologies: new chemicalpossibilities,” Indian Journal of Chemistry 48B:1721-1726 (2009).Alternatively, the polynucleic acid molecule is produced biologicallyusing an expression vector into which a polynucleic acid molecule hasbeen subcloned in an antisense orientation (i.e., RNA transcribed fromthe inserted polynucleic acid molecule will be of an antisenseorientation to a target polynucleic acid molecule of interest).

In some embodiments, a polynucleic acid molecule is synthesized via atandem synthesis methodology, wherein both strands are synthesized as asingle contiguous oligonucleotide fragment or strand separated by acleavable linker which is subsequently cleaved to provide separatefragments or strands that hybridize and permit purification of theduplex.

In some instances, a polynucleic acid molecule is also assembled fromtwo distinct nucleic acid strands or fragments wherein one fragmentincludes the sense region and the second fragment includes the antisenseregion of the molecule.

Additional modification methods for incorporating, for example, sugar,base and phosphate modifications include: Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344,565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al., U.S.Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al.,1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev.Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5,1999-2010. Such publications describe general methods and strategies todetermine the location of incorporation of sugar, base and/or phosphatemodifications and the like into nucleic acid molecules withoutmodulating catalysis.

In some instances, while chemical modification of the polynucleic acidmolecule internucleotide linkages with phosphorothioate,phosphorodithioate, and/or 5′-methylphosphonate linkages improvesstability, excessive modifications sometimes cause toxicity or decreasedactivity. Therefore, when designing nucleic acid molecules, the amountof these internucleotide linkages in some cases is minimized. In suchcases, the reduction in the concentration of these linkages lowerstoxicity, increases efficacy and higher specificity of these molecules.

Polynucleic Acid Molecule Conjugates

In some embodiments, a polynucleic acid molecule is further conjugatedto a polypeptide A for delivery to a site of interest. In some cases, apolynucleic acid molecule is conjugated to a polypeptide A andoptionally a polymeric moiety.

In some instances, at least one polypeptide A is conjugated to at leastone B. In some instances, the at least one polypeptide A is conjugatedto the at least one B to form an A-B conjugate. In some embodiments, atleast one A is conjugated to the 5′ terminus of B, the 3′ terminus of B,an internal site on B, or in any combinations thereof. In someinstances, the at least one polypeptide A is conjugated to at least twoB. In some instances, the at least one polypeptide A is conjugated to atleast 2, 3, 4, 5, 6, 7, 8, or more B.

In some embodiments, at least one polypeptide A is conjugated at oneterminus of at least one B while at least one C is conjugated at theopposite terminus of the at least one B to form an A-B-C conjugate. Insome instances, at least one polypeptide A is conjugated at one terminusof the at least one B while at least one of C is conjugated at aninternal site on the at least one B. In some instances, at least onepolypeptide A is conjugated directly to the at least one C. In someinstances, the at least one B is conjugated indirectly to the at leastone polypeptide A via the at least one C to form an A-C-B conjugate.

In some instances, at least one B and/or at least one C, and optionallyat least one D are conjugated to at least one polypeptide A. In someinstances, the at least one B is conjugated at a terminus (e.g., a 5′terminus or a 3′ terminus) to the at least one polypeptide A or areconjugated via an internal site to the at least one polypeptide A. Insome cases, the at least one C is conjugated either directly to the atleast one polypeptide A or indirectly via the at least one B. Ifindirectly via the at least one B, the at least one C is conjugatedeither at the same terminus as the at least one polypeptide A on B, atopposing terminus from the at least one polypeptide A, or independentlyat an internal site. In some instances, at least one additionalpolypeptide A is further conjugated to the at least one polypeptide A,to B, or to C. In additional instances, the at least one D is optionallyconjugated either directly or indirectly to the at least one polypeptideA, to the at least one B, or to the at least one C. If directly to theat least one polypeptide A, the at least one D is also optionallyconjugated to the at least one B to form an A-D-B conjugate or isoptionally conjugated to the at least one B and the at least one C toform an A-D-B-C conjugate. In some instances, the at least one D isdirectly conjugated to the at least one polypeptide A and indirectly tothe at least one B and the at least one C to form a D-A-B-C conjugate.If indirectly to the at least one polypeptide A, the at least one D isalso optionally conjugated to the at least one B to form an A-B-Dconjugate or is optionally conjugated to the at least one B and the atleast one C to form an A-B-D-C conjugate. In some instances, at leastone additional D is further conjugated to the at least one polypeptideA, to B, or to C.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19A.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19B.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19C.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19D.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19E.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19F.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19G.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19H.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19I.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19J.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19K.

In some embodiments, a polynucleic acid molecule conjugate comprises aconstruct as illustrated in FIG. 19L

The antibody cartoon as illustrated in FIG. 19M is for representationpurposes only and encompasses a humanized antibody or binding fragmentthereof, chimeric antibody or binding fragment thereof, monoclonalantibody or binding fragment thereof, monovalent Fab′, divalent Fab2,single-chain variable fragment (scFv), diabody, minibody, nanobody,single-domain antibody (sdAb), or camelid antibody or binding fragmentthereof.

Binding Moiety

In some embodiments, the binding moiety A is a polypeptide. In someinstances, the polypeptide is an antibody or its fragment thereof. Insome cases, the fragment is a binding fragment. In some instances, theantibody or binding fragment thereof comprises a humanized antibody orbinding fragment thereof, murine antibody or binding fragment thereof,chimeric antibody or binding fragment thereof, monoclonal antibody orbinding fragment thereof, monovalent Fab′, divalent Fab₂, F(ab)′₃fragments, single-chain variable fragment (scFv), bis-scFv, (scFv)₂,diabody, minibody, nanobody, triabody, tetrabody, disulfide stabilizedFv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelidantibody or binding fragment thereof, bispecific antibody or bidingfragment thereof, or a chemically modified derivative thereof.

In some instances, A is an antibody or binding fragment thereof. In someinstances, A is a humanized antibody or binding fragment thereof, murineantibody or binding fragment thereof, chimeric antibody or bindingfragment thereof, monoclonal antibody or binding fragment thereof,monovalent Fab′, divalent Fab₂, F(ab)′₃ fragments, single-chain variablefragment (scFv), bis-scFv, (scFv)₂, diabody, minibody, nanobody,triabody, tetrabody, disulfide stabilized Fv protein (“dsFv”),single-domain antibody (sdAb), Ig NAR, camelid antibody or bindingfragment thereof, bispecific antibody or biding fragment thereof, or achemically modified derivative thereof. In some instances, A is ahumanized antibody or binding fragment thereof. In some instances, A isa murine antibody or binding fragment thereof. In some instances, A is achimeric antibody or binding fragment thereof. In some instances, A is amonoclonal antibody or binding fragment thereof. In some instances, A isa monovalent Fab′. In some instances, A is a diavalent Fab₂. In someinstances, A is a single-chain variable fragment (scFv).

In some embodiments, the binding moiety A is a bispecific antibody orbinding fragment thereof. In some instances, the bispecific antibody isa trifunctional antibody or a bispecific mini-antibody. In some cases,the bispecific antibody is a trifunctional antibody. In some instances,the trifunctional antibody is a full length monoclonal antibodycomprising binding sites for two different antigens.

In some cases, the bispecific antibody is a bispecific mini-antibody. Insome instances, the bispecific mini-antibody comprises divalent Fab₂,F(ab)′₃ fragments, bis-scFv, (scFv)₂, diabody, minibody, triabody,tetrabody or a bi-specific T-cell engager (BiTE). In some embodiments,the bi-specific T-cell engager is a fusion protein that contains twosingle-chain variable fragments (scFvs) in which the two scFvs targetepitopes of two different antigens.

In some embodiments, the binding moiety A is a bispecific mini-antibody.In some instances, A is a bispecific Fab₂. In some instances, A is abispecific F(ab)′₃ fragment. In some cases, A is a bispecific bis-scFv.In some cases, A is a bispecific (scFv)₂. In some embodiments, A is abispecific diabody. In some embodiments, A is a bispecific minibody. Insome embodiments, A is a bispecific triabody. In other embodiments, A isa bispecific tetrabody. In other embodiments, A is a bi-specific T-cellengager (BiTE).

In some embodiments, the binding moiety A is a trispecific antibody. Insome instances, the trispecific antibody comprises F(ab)′₃ fragments ora triabody. In some instances, A is a trispecific F(ab)′₃ fragment. Insome cases, A is a triabody. In some embodiments, A is a trispecificantibody as described in Dimas, et al., “Development of a trispecificantibody designed to simultaneously and efficiently target threedifferent antigens on tumor cells,” Mol. Pharmaceutics, 12(9): 3490-3501(2015).

In some embodiments, the binding moiety A is an antibody or bindingfragment thereof that recognizes a cell surface protein. In someinstances, the binding moiety A is an antibody or binding fragmentthereof that recognizes a cell surface protein on a muscle cell. In somecases, the binding moiety A is an antibody or binding fragment thereofthat recognizes a cell surface protein on a skeletal muscle cell.

In some embodiments, exemplary antibodies include, but are not limitedto, an anti-myosin antibody, an anti-transferrin antibody, and anantibody that recognizes Muscle-Specific kinase (MuSK). In someinstances, the antibody is an anti-transferrin (anti-CD71) antibody.

In some embodiments, the binding moiety A is conjugated to a polynucleicacid molecule (B) non-specifically. In some instances, the bindingmoiety A is conjugated to a polynucleic acid molecule (B) via a lysineresidue or a cysteine residue, in a non-site specific manner. In someinstances, the binding moiety A is conjugated to a polynucleic acidmolecule (B) via a lysine residue in a non-site specific manner. In somecases, the binding moiety A is conjugated to a polynucleic acid molecule(B) via a cysteine residue in a non-site specific manner.

In some embodiments, the binding moiety A is conjugated to a polynucleicacid molecule (B) in a site-specific manner. In some instances, thebinding moiety A is conjugated to a polynucleic acid molecule (B)through a lysine residue, a cysteine residue, at the 5′-terminus, at the3′-terminus, an unnatural amino acid, or an enzyme-modified orenzyme-catalyzed residue, via a site-specific manner. In some instances,the binding moiety A is conjugated to a polynucleic acid molecule (B)through a lysine residue via a site-specific manner. In some instances,the binding moiety A is conjugated to a polynucleic acid molecule (B)through a cysteine residue via a site-specific manner. In someinstances, the binding moiety A is conjugated to a polynucleic acidmolecule (B) at the 5′-terminus via a site-specific manner. In someinstances, the binding moiety A is conjugated to a polynucleic acidmolecule (B) at the 3′-terminus via a site-specific manner. In someinstances, the binding moiety A is conjugated to a polynucleic acidmolecule (B) through an unnatural amino acid via a site-specific manner.In some instances, the binding moiety A is conjugated to a polynucleicacid molecule (B) through an enzyme-modified or enzyme-catalyzed residuevia a site-specific manner.

In some embodiments, one or more polynucleic acid molecule (B) isconjugated to a binding moiety A. In some instances, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 1 polynucleic acid molecule is conjugated to one binding moiety A.In some instances, about 2 polynucleic acid molecules are conjugated toone binding moiety A. In some instances, about 3 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 4 polynucleic acid molecules are conjugated to one binding moietyA. In some instances, about 5 polynucleic acid molecules are conjugatedto one binding moiety A. In some instances, about 6 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 7 polynucleic acid molecules are conjugated to one binding moietyA. In some instances, about 8 polynucleic acid molecules are conjugatedto one binding moiety A. In some instances, about 9 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 10 polynucleic acid molecules are conjugated to one binding moietyA. In some instances, about 11 polynucleic acid molecules are conjugatedto one binding moiety A. In some instances, about 12 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 13 polynucleic acid molecules are conjugated to one binding moietyA. In some instances, about 14 polynucleic acid molecules are conjugatedto one binding moiety A. In some instances, about 15 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 16 polynucleic acid molecules are conjugated to one binding moietyA. In some cases, the one or more polynucleic acid molecules are thesame. In other cases, the one or more polynucleic acid molecules aredifferent.

In some embodiments, the number of polynucleic acid molecule (B)conjugated to a binding moiety A forms a ratio. In some instances, theratio is referred to as a DAR (drug-to-antibody) ratio, in which thedrug as referred to herein is the polynucleic acid molecule (B). In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,or greater. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 1 or greater. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 2 or greater. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 3 or greater.In some instances, the DAR ratio of the polynucleic acid molecule (B) tobinding moiety A is about 4 or greater. In some instances, the DAR ratioof the polynucleic acid molecule (B) to binding moiety A is about 5 orgreater. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 6 or greater. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 7 or greater. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 8 or greater.In some instances, the DAR ratio of the polynucleic acid molecule (B) tobinding moiety A is about 9 or greater. In some instances, the DAR ratioof the polynucleic acid molecule (B) to binding moiety A is about 10 orgreater. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 11 or greater. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 12 or greater.

In some instances, the DAR ratio of the polynucleic acid molecule (B) tobinding moiety A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, or 16. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 1. In some instances, the DARratio of the polynucleic acid molecule (B) to binding moiety A is about2. In some instances, the DAR ratio of the polynucleic acid molecule (B)to binding moiety A is about 3. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 4. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 5. In some instances, the DAR ratio of the polynucleicacid molecule (B) to binding moiety A is about 6. In some instances, theDAR ratio of the polynucleic acid molecule (B) to binding moiety A isabout 7. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 8. In some instances, the DARratio of the polynucleic acid molecule (B) to binding moiety A is about9. In some instances, the DAR ratio of the polynucleic acid molecule (B)to binding moiety A is about 10. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 11. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 12. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 13. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 14. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 15. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 16.

In some instances, the DAR ratio of the polynucleic acid molecule (B) tobinding moiety A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,or 16. In some instances, the DAR ratio of the polynucleic acid molecule(B) to binding moiety A is 1. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is 2. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is 4. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is 6. In some instances, the DAR ratioof the polynucleic acid molecule (B) to binding moiety A is 8. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is 12.

In some instances, a conjugate comprising polynucleic acid molecule (B)and binding moiety A has improved activity as compared to a conjugatecomprising polynucleic acid molecule (B) without a binding moiety A. Insome instances, improved activity results in enhanced biologicallyrelevant functions, e.g., improved stability, affinity, binding,functional activity, and efficacy in treatment or prevention of adisease state. In some instances, the disease state is a result of oneor more mutated exons of a gene. In some instances, the conjugatecomprising polynucleic acid molecule (B) and binding moiety A results inincreased exon skipping of the one or more mutated exons as compared tothe conjugate comprising polynucleic acid molecule (B) without a bindingmoiety A. In some instances, exon skipping is increased by at least orabout 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or morethan 95% in the conjugate comprising polynucleic acid molecule (B) andbinding moiety A as compared to the conjugate comprising polynucleicacid molecule (B) without a binding moiety A.

In some embodiments, an antibody or its binding fragment is furthermodified using conventional techniques known in the art, for example, byusing amino acid deletion, insertion, substitution, addition, and/or byrecombination and/or any other modification (e.g. posttranslational andchemical modifications, such as glycosylation and phosphorylation) knownin the art either alone or in combination. In some instances, themodification further comprises a modification for modulating interactionwith Fc receptors. In some instances, the one or more modificationsinclude those described in, for example, International Publication No.WO97/34631, which discloses amino acid residues involved in theinteraction between the Fe domain and the FcRn receptor. Methods forintroducing such modifications in the nucleic acid sequence underlyingthe amino acid sequence of an antibody or its binding fragment is wellknown to the person skilled in the art.

In some instances, an antibody binding fragment further encompasses itsderivatives and includes polypeptide sequences containing at least oneCDR.

In some instances, the term “single-chain” as used herein means that thefirst and second domains of a bi-specific single chain construct arecovalently linked, preferably in the form of a co-linear amino acidsequence encodable by a single nucleic acid molecule.

In some instances, a bispecific single chain antibody construct relatesto a construct comprising two antibody derived binding domains. In suchembodiments, bi-specific single chain antibody construct is tandembi-scFv or diabody. In some instances, a scFv contains a VH and VLdomain connected by a linker peptide. In some instances, linkers are ofa length and sequence sufficient to ensure that each of the first andsecond domains can, independently from one another, retain theirdifferential binding specificities.

In some embodiments, binding to or interacting with as used hereindefines a binding/interaction of at least two antigen-interaction-siteswith each other. In some instances, antigen-interaction-site defines amotif of a polypeptide that shows the capacity of specific interactionwith a specific antigen or a specific group of antigens. In some cases,the binding/interaction is also understood to define a specificrecognition. In such cases, specific recognition refers to that theantibody or its binding fragment is capable of specifically interactingwith and/or binding to at least two amino acids of each of a targetmolecule. For example, specific recognition relates to the specificityof the antibody molecule, or to its ability to discriminate between thespecific regions of a target molecule. In additional instances, thespecific interaction of the antigen-interaction-site with its specificantigen results in an initiation of a signal, e.g. due to the inductionof a change of the conformation of the antigen, an oligomerization ofthe antigen, etc. In further embodiments, the binding is exemplified bythe specificity of a “key-lock-principle”. Thus in some instances,specific motifs in the amino acid sequence of theantigen-interaction-site and the antigen bind to each other as a resultof their primary, secondary or tertiary structure as well as the resultof secondary modifications of said structure. In such cases, thespecific interaction of the antigen-interaction-site with its specificantigen results as well in a simple binding of the site to the antigen.

In some instances, specific interaction further refers to a reducedcross-reactivity of the antibody or its binding fragment or a reducedoff-target effect. For example, the antibody or its binding fragmentthat bind to the polypeptide/protein of interest but do not or do notessentially bind to any of the other polypeptides are considered asspecific for the polypeptide/protein of interest. Examples for thespecific interaction of an antigen-interaction-site with a specificantigen comprise the specificity of a ligand for its receptor, forexample, the interaction of an antigenic determinant (epitope) with theantigenic binding site of an antibody.

Additional Binding Moieties

In some embodiments, the binding moiety is a plasma protein. In someinstances, the plasma protein comprises albumin. In some instances, thebinding moiety A is albumin. In some instances, albumin is conjugated byone or more of a conjugation chemistry described herein to a polynucleicacid molecule. In some instances, albumin is conjugated by nativeligation chemistry to a polynucleic acid molecule. In some instances,albumin is conjugated by lysine conjugation to a polynucleic acidmolecule.

In some instances, the binding moiety is a steroid. Exemplary steroidsinclude cholesterol, phospholipids, di- and triacylglycerols, fattyacids, hydrocarbons that are saturated, unsaturated, comprisesubstitutions, or combinations thereof. In some instances, the steroidis cholesterol. In some instances, the binding moiety is cholesterol. Insome instances, cholesterol is conjugated by one or more of aconjugation chemistry described herein to a polynucleic acid molecule.In some instances, cholesterol is conjugated by native ligationchemistry to a polynucleic acid molecule. In some instances, cholesterolis conjugated by lysine conjugation to a polynucleic acid molecule.

In some instances, the binding moiety is a polymer, including but notlimited to polynucleic acid molecule aptamers that bind to specificsurface markers on cells. In this instance the binding moiety is apolynucleic acid that does not hybridize to a target gene or mRNA, butinstead is capable of selectively binding to a cell surface markersimilarly to an antibody binding to its specific epitope of a cellsurface marker.

In some cases, the binding moiety is a peptide. In some cases, thepeptide comprises between about 1 and about 3 kDa. In some cases, thepeptide comprises between about 1.2 and about 2.8 kDa, about 1.5 andabout 2.5 kDa, or about 1.5 and about 2 kDa. In some instances, thepeptide is a bicyclic peptide. In some cases, the bicyclic peptide is aconstrained bicyclic peptide. In some instances, the binding moiety is abicyclic peptide (e.g., bicycles from Bicycle Therapeutics).

In additional cases, the binding moiety is a small molecule. In someinstances, the small molecule is an antibody-recruiting small molecule.In some cases, the antibody-recruiting small molecule comprises atarget-binding terminus and an antibody-binding terminus, in which thetarget-binding terminus is capable of recognizing and interacting with acell surface receptor. For example, in some instances, thetarget-binding terminus comprising a glutamate urea compound enablesinteraction with PSMA, thereby, enhances an antibody interaction with acell that expresses PSMA. In some instances, a binding moiety is a smallmolecule described in Zhang et al., “A remote arene-binding site onprostate specific membrane antigen revealed by antibody-recruiting smallmolecules,” J Am Chem Soc. 132(36): 12711-12716 (2010); or McEnaney, etal., “Antibody-recruiting molecules: an emerging paradigm for engagingimmune function in treating human disease,” ACS Chem Biol. 7(7):1139-1151 (2012).

Production of Antibodies or Binding Fragments Thereof

In some embodiments, polypeptides described herein (e.g., antibodies andits binding fragments) are produced using any method known in the art tobe useful for the synthesis of polypeptides (e.g., antibodies), inparticular, by chemical synthesis or by recombinant expression, and arepreferably produced by recombinant expression techniques.

In some instances, an antibody or its binding fragment thereof isexpressed recombinantly, and the nucleic acid encoding the antibody orits binding fragment is assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., 1994,BioTechniques 17:242), which involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligation of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a nucleic acid molecule encoding an antibody isoptionally generated from a suitable source (e.g., an antibody cDNAlibrary, or cDNA library generated from any tissue or cells expressingthe immunoglobulin) by PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence.

In some instances, an antibody or its binding is optionally generated byimmunizing an animal, such as a rabbit, to generate polyclonalantibodies or, more preferably, by generating monoclonal antibodies,e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or,as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole etal. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). Alternatively, a clone encoding at least the Fabportion of the antibody is optionally obtained by screening Fabexpression libraries (e.g., as described in Huse et al., 1989, Science246:1275-1281) for clones of Fab fragments that bind the specificantigen or by screening antibody libraries (See, e.g., Clackson et al.,1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA94:4937).

In some embodiments, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al.,1985, Nature 314:452-454) by splicing genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity are used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region, e.g., humanized antibodies.

In some embodiments, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are adapted toproduce single chain antibodies. Single chain antibodies are formed bylinking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide. Techniquesfor the assembly of functional Fv fragments in E. coli are alsooptionally used (Skerra et al., 1988, Science 242:1038-1041).

In some embodiments, an expression vector comprising the nucleotidesequence of an antibody or the nucleotide sequence of an antibody istransferred to a host cell by conventional techniques (e.g.,electroporation, liposomal transfection, and calcium phosphateprecipitation), and the transfected cells are then cultured byconventional techniques to produce the antibody. In specificembodiments, the expression of the antibody is regulated by aconstitutive, an inducible or a tissue, specific promoter.

In some embodiments, a variety of host-expression vector systems isutilized to express an antibody or its binding fragment describedherein. Such host-expression systems represent vehicles by which thecoding sequences of the antibody is produced and subsequently purified,but also represent cells that are, when transformed or transfected withthe appropriate nucleotide coding sequences, express an antibody or itsbinding fragment in situ. These include, but are not limited to,microorganisms such as bacteria (e.g., E. coli and B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing an antibody or its binding fragmentcoding sequences; yeast (e.g., Saccharomyces Pichia) transformed withrecombinant yeast expression vectors containing an antibody or itsbinding fragment coding sequences; insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus) containing anantibody or its binding fragment coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containing anantibody or its binding fragment coding sequences; or mammalian cellsystems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5Kpromoter).

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. In some instances, cell lines that stablyexpress an antibody are optionally engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellsare transformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells are thenallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci that in turnare cloned and expanded into cell lines. This method can advantageouslybe used to engineer cell lines which express the antibody or its bindingfragments.

In some instances, a number of selection systems are used, including butnot limited to the herpes simplex virus thymidine kinase (Wigler et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), andadenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genesare employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance are used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan,1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev.Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215) and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds., 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY;and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, CurrentProtocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin etal., 1981, J. Mol. Biol. 150:1).

In some instances, the expression levels of an antibody are increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing an antibodyis amplifiable, an increase in the level of inhibitor present in cultureof host cell will increase the number of copies of the marker gene.Since the amplified region is associated with the nucleotide sequence ofthe antibody, production of the antibody will also increase (Crouse etal., 1983, Mol. Cell Biol. 3:257).

In some instances, any method known in the art for purification oranalysis of an antibody or antibody conjugates is used, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Exemplarychromatography methods included, but are not limited to, strong anionexchange chromatography, hydrophobic interaction chromatography, sizeexclusion chromatography, and fast protein liquid chromatography.

Conjugation Chemistry

In some embodiments, a polynucleic acid molecule B is conjugated to abinding moiety. In some instances, the binding moiety comprises aminoacids, peptides, polypeptides, proteins, antibodies, antigens, toxins,hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates,polymers such as polyethylene glycol and polypropylene glycol, as wellas analogs or derivatives of all of these classes of substances.Additional examples of binding moiety also include steroids, such ascholesterol, phospholipids, di- and triacylglycerols, fatty acids,hydrocarbons (e.g., saturated, unsaturated, or contains substitutions),enzyme substrates, biotin, digoxigenin, and polysaccharides. In someinstances, the binding moiety is an antibody or binding fragmentthereof. In some instances, the polynucleic acid molecule is furtherconjugated to a polymer, and optionally an endosomolytic moiety.

In some embodiments, the polynucleic acid molecule is conjugated to thebinding moiety by a chemical ligation process. In some instances, thepolynucleic acid molecule is conjugated to the binding moiety by anative ligation. In some instances, the conjugation is as described in:Dawson, et al. “Synthesis of proteins by native chemical ligation,”Science 1994, 266, 776-779; Dawson, et al. “Modulation of Reactivity inNative Chemical Ligation through the Use of Thiol Additives,” J. Am.Chem. Soc. 1997, 119, 4325-4329; Hackeng, et al. “Protein synthesis bynative chemical ligation: Expanded scope by using straightforwardmethodology.,” Proc. Natl. Acad. Sci. USA 1999, 96, 10068-10073; or Wu,et al. “Building complex glycopeptides: Development of a cysteine-freenative chemical ligation protocol,” Angew. Chem. Int. Ed. 2006, 45,4116-4125. In some instances, the conjugation is as described in U.S.Pat. No. 8,936,910. In some embodiments, the polynucleic acid moleculeis conjugated to the binding moiety either site-specifically ornon-specifically via native ligation chemistry.

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a site-directed method utilizing a “traceless”coupling technology (Philochem). In some instances, the “traceless”coupling technology utilizes an N-terminal 1,2-aminothiol group on thebinding moiety which is then conjugate with a polynucleic acid moleculecontaining an aldehyde group. (see Casi et al., “Site-specific tracelesscoupling of potent cytotoxic drugs to recombinant antibodies forpharmacodelivery,” JACS 134(13): 5887-5892 (2012))

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a site-directed method utilizing an unnatural aminoacid incorporated into the binding moiety. In some instances, theunnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In someinstances, the keto group of pAcPhe is selectively coupled to analkoxy-amine derivatived conjugating moiety to form an oxime bond. (seeAxup et al., “Synthesis of site-specific antibody-drug conjugates usingunnatural amino acids,” PNAS 109(40): 16101-16106 (2012)).

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a site-directed method utilizing an enzyme-catalyzedprocess. In some instances, the site-directed method utilizes SMARTag™technology (Catalent, Inc.). In some instances, the SMARTag™ technologycomprises generation of a formylglycine (FGly) residue from cysteine byformylglycine-generating enzyme (FGE) through an oxidation process underthe presence of an aldehyde tag and the subsequent conjugation of FGlyto an alkylhydraine-functionalized polynucleic acid molecule viahydrazino-Pictet-Spengler (HIPS) ligation. (see Wu et al.,“Site-specific chemical modification of recombinant proteins produced inmammalian cells by using the genetically encoded aldehyde tag,” PNAS106(9): 3000-3005 (2009); Agarwal, et al., “A Pictet-Spengler ligationfor protein chemical modification,” PNAS 110(1): 46-51 (2013))

In some instances, the enzyme-catalyzed process comprises microbialtransglutaminase (mTG). In some cases, the polynucleic acid molecule isconjugated to the binding moiety utilizing a microbialtransglutaminase-catalyzed process. In some instances, mTG catalyzes theformation of a covalent bond between the amide side chain of a glutaminewithin the recognition sequence and a primary amine of a functionalizedpolynucleic acid molecule. In some instances, mTG is produced fromStreptomyces mobarensis. (see Strop et al., “Location matters: site ofconjugation modulates stability and pharmacokinetics of antibody drugconjugates,” Chemistry and Biology 20(2) 161-167 (2013))

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a method as described in PCT Publication No.WO2014/140317, which utilizes a sequence-specific transpeptidase.

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a method as described in U.S. Patent Publication Nos.2015/0105539 and 2015/0105540.

Polymer Conjugating Moiety

In some embodiments, a polymer moiety C is further conjugated to apolynucleic acid molecule described herein, a binding moiety describedherein, or in combinations thereof. In some instances, a polymer moietyC is conjugated a polynucleic acid molecule. In some cases, a polymermoiety C is conjugated to a binding moiety. In other cases, a polymermoiety C is conjugated to a polynucleic acid molecule-binding moietymolecule. In additional cases, a polymer moiety C is conjugated, asillustrated supra.

In some instances, the polymer moiety C is a natural or syntheticpolymer, consisting of long chains of branched or unbranched monomers,and/or cross-linked network of monomers in two or three dimensions. Insome instances, the polymer moiety C includes a polysaccharide, lignin,rubber, or polyalkylen oxide (e.g., polyethylene glycol). In someinstances, the at least one polymer moiety C includes, but is notlimited to, alpha-, omega-dihydroxylpolyethyleneglycol, biodegradablelactone-based polymer, e.g. polyacrylic acid, polylactide acid (PLA),poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin,polyamide, polycyanoacrylate, polyimide, polyethylene terephthalate(also known as poly(ethylene terephthalate), PET, PETG, or PETE),polytetramethylene glycol (PTG), or polyurethane as well as mixturesthereof. As used herein, a mixture refers to the use of differentpolymers within the same compound as well as in reference to blockcopolymers. In some cases, block copolymers are polymers wherein atleast one section of a polymer is build up from monomers of anotherpolymer. In some instances, the polymer moiety C comprises polyalkyleneoxide. In some instances, the polymer moiety C comprises PEG. In someinstances, the polymer moiety C comprises polyethylene imide (PEI) orhydroxy ethyl starch (HES).

In some instances, C is a PEG moiety. In some instances, the PEG moietyis conjugated at the 5′ terminus of the polynucleic acid molecule whilethe binding moiety is conjugated at the 3′ terminus of the polynucleicacid molecule. In some instances, the PEG moiety is conjugated at the 3′terminus of the polynucleic acid molecule while the binding moiety isconjugated at the 5′ terminus of the polynucleic acid molecule. In someinstances, the PEG moiety is conjugated to an internal site of thepolynucleic acid molecule. In some instances, the PEG moiety, thebinding moiety, or a combination thereof, are conjugated to an internalsite of the polynucleic acid molecule. In some instances, theconjugation is a direct conjugation. In some instances, the conjugationis via native ligation.

In some embodiments, the polyalkylene oxide (e.g., PEG) is apolydisperse or monodisperse compound. In some instances, polydispersematerial comprises disperse distribution of different molecular weightof the material, characterized by mean weight (weight average) size anddispersity. In some instances, the monodisperse PEG comprises one sizeof molecules. In some embodiments, C is poly- or monodispersedpolyalkylene oxide (e.g., PEG) and the indicated molecular weightrepresents an average of the molecular weight of the polyalkylene oxide,e.g., PEG, molecules.

In some embodiments, the molecular weight of the polyalkylene oxide(e.g., PEG) is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750,4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000,10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.

In some embodiments, C is polyalkylene oxide (e.g., PEG) and has amolecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100,2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500,3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500,8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000Da. In some embodiments, C is PEG and has a molecular weight of about200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500,2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500,4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000,20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In someinstances, the molecular weight of C is about 200 Da. In some instances,the molecular weight of C is about 300 Da. In some instances, themolecular weight of C is about 400 Da. In some instances, the molecularweight of C is about 500 Da. In some instances, the molecular weight ofC is about 600 Da. In some instances, the molecular weight of C is about700 Da. In some instances, the molecular weight of C is about 800 Da. Insome instances, the molecular weight of C is about 900 Da. In someinstances, the molecular weight of C is about 1000 Da. In someinstances, the molecular weight of C is about 1100 Da. In someinstances, the molecular weight of C is about 1200 Da. In someinstances, the molecular weight of C is about 1300 Da. In someinstances, the molecular weight of C is about 1400 Da. In someinstances, the molecular weight of C is about 1450 Da. In someinstances, the molecular weight of C is about 1500 Da. In someinstances, the molecular weight of C is about 1600 Da. In someinstances, the molecular weight of C is about 1700 Da. In someinstances, the molecular weight of C is about 1800 Da. In someinstances, the molecular weight of C is about 1900 Da. In someinstances, the molecular weight of C is about 2000 Da. In someinstances, the molecular weight of C is about 2100 Da. In someinstances, the molecular weight of C is about 2200 Da. In someinstances, the molecular weight of C is about 2300 Da. In someinstances, the molecular weight of C is about 2400 Da. In someinstances, the molecular weight of C is about 2500 Da. In someinstances, the molecular weight of C is about 2600 Da. In someinstances, the molecular weight of C is about 2700 Da. In someinstances, the molecular weight of C is about 2800 Da. In someinstances, the molecular weight of C is about 2900 Da. In someinstances, the molecular weight of C is about 3000 Da. In someinstances, the molecular weight of C is about 3250 Da. In someinstances, the molecular weight of C is about 3350 Da. In someinstances, the molecular weight of C is about 3500 Da. In someinstances, the molecular weight of C is about 3750 Da. In someinstances, the molecular weight of C is about 4000 Da. In someinstances, the molecular weight of C is about 4250 Da. In someinstances, the molecular weight of C is about 4500 Da. In someinstances, the molecular weight of C is about 4600 Da. In someinstances, the molecular weight of C is about 4750 Da. In someinstances, the molecular weight of C is about 5000 Da. In someinstances, the molecular weight of C is about 5500 Da. In someinstances, the molecular weight of C is about 6000 Da. In someinstances, the molecular weight of C is about 6500 Da. In someinstances, the molecular weight of C is about 7000 Da. In someinstances, the molecular weight of C is about 7500 Da. In someinstances, the molecular weight of C is about 8000 Da. In someinstances, the molecular weight of C is about 10,000 Da. In someinstances, the molecular weight of C is about 12,000 Da. In someinstances, the molecular weight of C is about 20,000 Da. In someinstances, the molecular weight of C is about 35,000 Da. In someinstances, the molecular weight of C is about 40,000 Da. In someinstances, the molecular weight of C is about 50,000 Da. In someinstances, the molecular weight of C is about 60,000 Da. In someinstances, the molecular weight of C is about 100,000 Da.

In some embodiments, the polyalkylene oxide (e.g., PEG) comprisesdiscrete ethylene oxide units (e.g., four to about 48 ethylene oxideunits). In some instances, the polyalkylene oxide comprising thediscrete ethylene oxide units is a linear chain. In other cases, thepolyalkylene oxide comprising the discrete ethylene oxide units is abranched chain.

In some instances, the polymer moiety C is a polyalkylene oxide (e.g.,PEG) comprising discrete ethylene oxide units. In some cases, thepolymer moiety C comprises between about 4 and about 48 ethylene oxideunits. In some cases, the polymer moiety C comprises about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, about 15, about 16, about 17, about 18, about 19, about20, about 21, about 22, about 23, about 24, about 25, about 26, about27, about 28, about 29, about 30, about 31, about 32, about 33, about34, about 35, about 36, about 37, about 38, about 39, about 40, about41, about 42, about 43, about 44, about 45, about 46, about 47, or about48 ethylene oxide units.

In some instances, the polymer moiety C is a discrete PEG comprising,e.g., between about 4 and about 48 ethylene oxide units. In some cases,the polymer moiety C is a discrete PEG comprising, e.g., about 4, about5, about 6, about 7, about 8, about 9, about 10, about 11, about 12,about 13, about 14, about 15, about 16, about 17, about 18, about 19,about 20, about 21, about 22, about 23, about 24, about 25, about 26,about 27, about 28, about 29, about 30, about 31, about 32, about 33,about 34, about 35, about 36, about 37, about 38, about 39, about 40,about 41, about 42, about 43, about 44, about 45, about 46, about 47, orabout 48 ethylene oxide units. In some cases, the polymer moiety C is adiscrete PEG comprising, e.g., about 4 ethylene oxide units. In somecases, the polymer moiety C is a discrete PEG comprising, e.g., about 5ethylene oxide units. In some cases, the polymer moiety C is a discretePEG comprising, e.g., about 6 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 7 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 8 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 9 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 10 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 11 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 12 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 13 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 14 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 15 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 16 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 17 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 18 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 19 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 20 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 21 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 22 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 23 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 24 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 25 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 26 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 27 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 28 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 29 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 30 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 31 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 32 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 33 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 34 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 35 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 36 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 37 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 38 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 39 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 40 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 41 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 42 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 43 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 44 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 45 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 46 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 47 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 48 ethylene oxide units.

In some cases, the polymer moiety C is dPEG® (Quanta Biodesign Ltd).

In some embodiments, the polymer moiety C comprises a cationic mucicacid-based polymer (cMAP). In some instances, cMAP comprises one or moresubunit of at least one repeating subunit, and the subunit structure isrepresented as Formula (V):

wherein m is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10, preferably 4-6 or 5; and n is independently at each occurrence 1,2, 3, 4, or 5. In some embodiments, m and n are, for example, about 10.

In some instances, cMAP is further conjugated to a PEG moiety,generating a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, ora cMAP-PEG-cMAP triblock polymer. In some instances, the PEG moiety isin a range of from about 500 Da to about 50,000 Da. In some instances,the PEG moiety is in a range of from about 500 Da to about 1000 Da,greater than 1000 Da to about 5000 Da, greater than 5000 Da to about10,000 Da, greater than 10,000 to about 25,000 Da, greater than 25,000Da to about 50,000 Da, or any combination of two or more of theseranges.

In some instances, the polymer moiety C is cMAP-PEG copolymer, anmPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. Insome cases, the polymer moiety C is cMAP-PEG copolymer. In other cases,the polymer moiety C is an mPEG-cMAP-PEGm triblock polymer. Inadditional cases, the polymer moiety C is a cMAP-PEG-cMAP triblockpolymer.

In some embodiments, the polymer moiety C is conjugated to thepolynucleic acid molecule, the binding moiety, and optionally to theendosomolytic moiety as illustrated supra.

Endosomolytic Moiety

In some embodiments, a molecule of Formula (I): A-X₁—B—X₂—C, furthercomprises an additional conjugating moiety. In some instances, theadditional conjugating moiety is an endosomolytic moiety. In some cases,the endosomolytic moiety is a cellular compartmental release component,such as a compound capable of releasing from any of the cellularcompartments known in the art, such as the endosome, lysosome,endoplasmic reticulum (ER), golgi apparatus, microtubule, peroxisome, orother vesicular bodies with the cell. In some cases, the endosomolyticmoiety comprises an endosomolytic polypeptide, an endosomolytic polymer,an endosomolytic lipid, or an endosomolytic small molecule. In somecases, the endosomolytic moiety comprises an endosomolytic polypeptide.In other cases, the endosomolytic moiety comprises an endosomolyticpolymer.

Endosomolytic Polypeptides

In some embodiments, a molecule of Formula (I): A-X₁—B—X₂—C, is furtherconjugated with an endosomolytic polypeptide. In some cases, theendosomolytic polypeptide is a pH-dependent membrane active peptide. Insome cases, the endosomolytic polypeptide is an amphipathic polypeptide.In additional cases, the endosomolytic polypeptide is a peptidomimetic.In some instances, the endosomolytic polypeptide comprises INF,melittin, meucin, or their respective derivatives thereof. In someinstances, the endosomolytic polypeptide comprises INF or itsderivatives thereof. In other cases, the endosomolytic polypeptidecomprises melittin or its derivatives thereof. In additional cases, theendosomolytic polypeptide comprises meucin or its derivatives thereof.

In some instances, INF7 is a 24 residue polypeptide those sequencecomprises CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO: 1), orGLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO: 2). In some instances, INF7 or itsderivatives comprise a sequence of:

(SEQ ID NO: 3) GLFEAIEGFIENGWEGMIWDYGSGSCG, (SEQ ID NO: 4)GLFEAIEGFIENGWEGMIDGWYG-(PEG)6-NH2, or (SEQ ID NO: 5)GLFEAIEGFIENGWEGMIWDYG-SGSC-K(Ga1NAc)2.

In some cases, melittin is a 26 residue polypeptide those sequencecomprises CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO: 6), orGIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 7). In some instances, melittincomprises a polypeptide sequence as described in U.S. Pat. No.8,501,930.

In some instances, meucin is an antimicrobial peptide (AMP) derived fromthe venom gland of the scorpion Mesobuthus eupeus. In some instances,meucin comprises of meucin-13 those sequence comprises IFGAIAGLLKNIF-NH₂(SEQ ID NO: 8) and meucin-18 those sequence comprises FFGHLFKLATKIIPSLFQ(SEQ ID NO: 9).

In some instances, the endosomolytic polypeptide comprises a polypeptidein which its sequence is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%sequence identity to INF7 or its derivatives thereof, melittin or itsderivatives thereof, or meucin or its derivatives thereof. In someinstances, the endosomolytic moiety comprises INF7 or its derivativesthereof, melittin or its derivatives thereof, or meucin or itsderivatives thereof.

In some instances, the endosomolytic moiety is INF7 or its derivativesthereof. In some cases, the endosomolytic moiety comprises a polypeptidehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1-5. In somecases, the endosomolytic moiety comprises a polypeptide having at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to SEQ ID NO: 1. In some cases, the endosomolyticmoiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 2-5. In some cases, the endosomolytic moiety comprises SEQID NO: 1. In some cases, the endosomolytic moiety comprises SEQ ID NO:2-5. In some cases, the endosomolytic moiety consists of SEQ ID NO: 1.In some cases, the endosomolytic moiety consists of SEQ ID NO: 2-5.

In some instances, the endosomolytic moiety is melittin or itsderivatives thereof. In some cases, the endosomolytic moiety comprises apolypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 6 or7. In some cases, the endosomolytic moiety comprises a polypeptidehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6. In some cases,the endosomolytic moiety comprises a polypeptide having at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO: 7. In some cases, the endosomolyticmoiety comprises SEQ ID NO: 6. In some cases, the endosomolytic moietycomprises SEQ ID NO: 7. In some cases, the endosomolytic moiety consistsof SEQ ID NO: 6. In some cases, the endosomolytic moiety consists of SEQID NO: 7.

In some instances, the endosomolytic moiety is meucin or its derivativesthereof. In some cases, the endosomolytic moiety comprises a polypeptidehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 8 or 9. In somecases, the endosomolytic moiety comprises a polypeptide having at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to SEQ ID NO: 8. In some cases, the endosomolyticmoiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 9. In some cases, the endosomolytic moiety comprises SEQID NO: 8. In some cases, the endosomolytic moiety comprises SEQ ID NO:9. In some cases, the endosomolytic moiety consists of SEQ ID NO: 8. Insome cases, the endosomolytic moiety consists of SEQ ID NO: 9.

In some instances, the endosomolytic moiety comprises a sequence asillustrated in

TABLE 1 AMINO ACID SEQ SE- ID NAME ORIGIN QUENCE NO: TYPE Pep-1 NLS fromKETW 10 Primary Simian Virus WETW amphipathic 40 large WTEW antigen andSQPK Reverse KKRKV transcriptase of HIV pVEC VE-cadherin LLII 11 PrimaryLRRR amphipathic RIRK QAHA HSK VT5 Synthetic DPKG 12 β-sheet peptideDPKG amphipathic VTVT VTVT VTGK GDPK PD C105Y 1- CSIP 13 — antitrypsinPEVK FNKP FVYL I Transportan Galanin and GWTL 14 Primary mastoparan NSAGamphipathic YLLG KINL KALA ALAK KIL TP10 Galanin and AGYL 15 Primarymastoparan LGKI amphipathic NLKA LAAL AKKI L MPG A hydrofobic GALF 16β-sheet domain LGFL amphipathic from the GAAG fusion STMG sequence Aof HIV gp41 and NLS of SV40 T antigen gH625 Glycoprotein HGLA 17Secondary gH of HSV STLT amphipathic type I RWAH α-helical YNAL IRAFCADY PPTGI GLWR 18 Secondary peptide ALWR amphipathic LLRS α-helicalLWRL LWRA GALA Synthetic WEAA 19 Secondary peptide LAEA amphipathic LAEAα-helical LAEH LAEA LAEA LEAL AA INF Influenza GLFE 20 SecondaryHA2 fusion AIEG amphipathic peptide FIEN α-helical/ GWEG pH- MIDGdependent WYGC membrane active peptide HA2E5- Influenza GLFG 21Secondary TAT HA2 AIAG amphipathic subunit of FIEN α-helical/ influenzaGWEG pH- virus X31 MIDG dependent strain WYG membrane fusion activepeptide peptide HA2- Influenza GLFG 22 pH- penetratin HA2 AIAG dependentsubunit of FIEN membrane influenza GWEG active virus X31 MIDG peptidestrain RQIK fusion IWFQ peptide NRRM KWKK -ami de HA-K4 Influenza GLFG23 pH- HA2 AIAG dependent subunit of FIEN membrane influenza GWEG activevirus X31 MIDG peptide strain -SSK fusion KKK peptide HA2E4 InfluenzaGLFE 24 pH- HA2 AIAG dependent subunit of FIEN membrane influenza GWEGactive virus X31 MIDG peptide strain GGYC fusion peptide H5WYG HA2 GLFH25 pH- analogue AIAH dependent FIHG membrane GWHG active LIHG peptideWYG GALA- INF3 fusion GLFE 26 pH- INF3- peptide AIEG dependent (PEG)6-NHFIEN membrane GWEG active LAEA peptide LAEA LEAL AA- (PEG)6- NH2 CM18-Cecropin-A- KWKL 27 pH- TAT11 Melittin₂₋₁₂ FKKI dependent (CM₁₈) GAVLmembrane fusion KVLT active peptide TG-Y peptide GRKK RRQR RR

In some cases, the endosomolytic moiety comprises a Bak BH3 polypeptidewhich induces apoptosis through antagonization of suppressor targetssuch as Bel-2 and/or Bcl-x_(L). In some instances, the endosomolyticmoiety comprises a Bak BH3 polypeptide described in Albarran, et al.,“Efficient intracellular delivery of a pro-apoptotic peptide with apH-responsive carrier,” Reactive & Functional Polymers 71: 261-265(2011).

In some instances, the endosomolytic moiety comprises a polypeptide(e.g., a cell-penetrating polypeptide) as described in PCT PublicationNos. WO2013/166155 or WO2015/069587.

Endosomolytic Lipids

In some embodiments, the endosomolytic moiety is a lipid (e.g., afusogenic lipid). In some embodiments, a molecule of Formula (I):A-X₁—B—X₂—C, is further conjugated with an endosomolytic lipid (e.g.,fusogenic lipid). Exemplary fusogenic lipids include1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine(POPE), palmitoyloleoylphosphatidylcholine (POPC),(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin),N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine(DLin-k-DMA) andN-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine(XTC).

In some instances, an endosomolytic moiety is a lipid (e.g., a fusogeniclipid) described in PCT Publication No. WO09/126,933.

Endosomolytic Small Molecules

In some embodiments, the endosomolytic moiety is a small molecule. Insome embodiments, a molecule of Formula (I): A-X₁—B—X₂—C, is furtherconjugated with an endosomolytic small molecule. Exemplary smallmolecules suitable as endosomolytic moieties include, but are notlimited to, quinine, chloroquine, hydroxychloroquines, amodiaquins(carnoquines), amopyroquines, primaquines, mefloquines, nivaquines,halofantrines, quinone imines, or a combination thereof. In someinstances, quinoline endosomolytic moieties include, but are not limitedto, 7-chloro-4-(4-diethylamino-1-methylbutyl-amino)quinoline(chloroquine);7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutyl-amino)quinoline(hydroxychloroquine);7-fluoro-4-(4-diethylamino-1-methylbutyl-amino)quinoline;4-(4-diethylamino-1-methylbutylamino) quinoline;7-hydroxy-4-(4-diethyl-amino-1-methylbutylamino)quinoline;7-chloro-4-(4-diethylamino-1-butylamino)quinoline(desmethylchloroquine);7-fluoro-4-(4-diethylamino-1-butylamino)quinoline);4-(4-diethyl-amino-1-butylamino)quinoline;7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-fluoro-4-(1-carboxy-4-diethyl-amino-1-butylamino)quinoline;4-(1-carboxy-4-diethylamino-1-butylamino) quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-fluoro-4-(1-carboxy-4-diethyl-amino-1-methylbutylamino)quinoline;4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;4-(4-ethyl-(2-hydroxy-ethyl)-amino-1-methylbutylamino-)quinoline;7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;hydroxychloroquine phosphate;7-chloro-4-(4-ethyl-(2-hydroxyethyl-1)-amino-1-butylamino)quinoline(desmethylhydroxychloroquine);7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino) quinoline;7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;8-[(4-aminopentyl)amino-6-methoxydihydrochloride quinoline;1-acetyl-1,2,3,4-tetrahydroquinoline;8-[(4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride;1-butyryl-1,2,3,4-tetrahydroquinoline;3-chloro-4-(4-hydroxy-alpha,alpha′-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline,4-[(4-diethyl-amino)-1-methylbutyl-amino]-6-methoxyquinoline;3-fluoro-4-(4-hydroxy-alpha,alpha′-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline,4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline;4-(4-hydroxy-alpha,alpha′-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline;4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline;3,4-dihydro-1-(2H)-quinolinecarboxyaldehyde; 1,1′-pentamethylenediquinoleinium diiodide; 8-quinolinol sulfate and amino, aldehyde,carboxylic, hydroxyl, halogen, keto, sulfhydryl and vinyl derivatives oranalogs thereof. In some instances, an endosomolytic moiety is a smallmolecule described in Naisbitt et al (1997, J Pharmacol Exp Therapy280:884-893) and in U.S. Pat. No. 5,736,557.

Linkers

In some embodiments, a linker described herein is a cleavable linker ora non-cleavable linker. In some instances, the linker is a cleavablelinker. In other instances, the linker is a non-cleavable linker.

In some cases, the linker is a non-polymeric linker. A non-polymericlinker refers to a linker that does not contain a repeating unit ofmonomers generated by a polymerization process. Exemplary non-polymericlinkers include, but are not limited to, C₁-C₆ alkyl group (e.g., a C₅,C₄, C₃, C₂, or C₁ alkyl group), homobifunctional cross linkers,heterobifunctional cross linkers, peptide linkers, traceless linkers,self-immolative linkers, maleimide-based linkers, or combinationsthereof. In some cases, the non-polymeric linker comprises a C₁-C₆ alkylgroup (e.g., a C₅, C₄, C₃, C₂, or C₁ alkyl group), a homobifunctionalcross linker, a heterobifunctional cross linker, a peptide linker, atraceless linker, a self-immolative linker, a maleimide-based linker, ora combination thereof. In additional cases, the non-polymeric linkerdoes not comprise more than two of the same type of linkers, e.g., morethan two homobifunctional cross linkers, or more than two peptidelinkers. In further cases, the non-polymeric linker optionally comprisesone or more reactive functional groups.

In some instances, the non-polymeric linker does not encompass a polymerthat is described above. In some instances, the non-polymeric linkerdoes not encompass a polymer encompassed by the polymer moiety C. Insome cases, the non-polymeric linker does not encompass a polyalkyleneoxide (e.g., PEG). In some cases, the non-polymeric linker does notencompass a PEG.

In some instances, the linker comprises a homobifunctional linker.Exemplary homobifunctional linkers include, but are not limited to,Lomant's reagent dithiobis (succinimidylpropionate) DSP,3′3′-dithiobis(sulfosuccinimidyl proprionate (DTSSP), disuccinimidylsuberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyltartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethyleneglycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG),N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA),dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS),dimethyl-3,3′-dithiobispropionimidate (DTBP),1,4-di-3′-(2′-pyridyldithio)propionamido)butane (DPDPB),bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), suchas e.g. 1,5-difluoro-2,4-dinitrobenzene or1,3-difluoro-4,6-dinitrobenzene, 4,4′-difluoro-3,3′-dinitrophenylsulfone(DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED),formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipicacid dihydrazide, carbohydrazide, o-toluidine, 3,3′-dimethylbenzidine,benzidine, α,α′-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid,N,N′-ethylene-bis(iodoacetamide), orN,N′-hexamethylene-bis(iodoacetamide).

In some embodiments, the linker comprises a heterobifunctional linker.Exemplary heterobifunctional linker include, but are not limited to,amine-reactive and sulfhydryl cross-linkers such as N-succinimidyl3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble-long-chainN-succinimidyl 3-(2-pyridyldithio) propionate (sulfo-LC-sPDP),succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT),sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate(sulfo-LC-sMPT),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC),sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs),m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs),N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB),sulfosuccinimidyl(4-iodoacteyl)aminobenzoate (sulfo-sIAB),succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB),sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB),N-(γ-maleimidobutyryloxy)succinimide ester (GMBs),N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs),succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC),succinimidyl6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino) hexanoate(sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive andsulfhydryl-reactive cross-linkers such as 4-(4-N-maleimidophenyl)butyricacid hydrazide (MPBH),4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M₂C₂H),3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive andphotoreactive cross-linkers such asN-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA),N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA),sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA),sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3′-dithiopropionate(sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB),N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB),N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sANPAH),sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate(sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs),sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiopropionate(sAND), N-succinimidyl-4(4-azidophenyl)1,3′-dithiopropionate (sADP),N-sulfosuccinimidyl(4-azidophenyl)-1,3′-dithiopropionate (sulfo-sADP),sulfosuccinimidyl 4-(p-azidophenyl)butyrate (sulfo-sAPB),sulfosuccinimidyl2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate(sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate(sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP),p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP),sulfhydryl-reactive and photoreactive cross-linkers suchasl-(p-Azidosalicylamido)-4-(iodoacetamido)butane (AsIB),N-[4-(p-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide(APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidecarbonyl-reactive and photoreactive cross-linkers such as p-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive cross-linkerssuch as 4-(p-azidosalicylamido)butylamine (AsBA), and arginine-reactiveand photoreactive cross-linkers such as p-azidophenyl glyoxal (APG).

In some instances, the linker comprises a reactive functional group. Insome cases, the reactive functional group comprises a nucleophilic groupthat is reactive to an electrophilic group present on a binding moiety.Exemplary electrophilic groups include carbonyl groups-such as aldehyde,ketone, carboxylic acid, ester, amide, enone, acyl halide or acidanhydride. In some embodiments, the reactive functional group isaldehyde. Exemplary nucleophilic groups include hydrazide, oxime, amino,hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

In some embodiments, the linker comprises a maleimide group. In someinstances, the maleimide group is also referred to as a maleimidespacer. In some instances, the maleimide group further encompasses acaproic acid, forming maleimidocaproyl (me). In some cases, the linkercomprises maleimidocaproyl (me). In some cases, the linker ismaleimidocaproyl (me). In other instances, the maleimide group comprisesa maleimidomethyl group, such assuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) orsulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-sMCC) described above.

In some embodiments, the maleimide group is a self-stabilizingmaleimide. In some instances, the self-stabilizing maleimide utilizesdiaminopropionic acid (DPR) to incorporate a basic amino group adjacentto the maleimide to provide intramolecular catalysis of tiosuccinimidering hydrolysis, thereby eliminating maleimide from undergoing anelimination reaction through a retro-Michael reaction. In someinstances, the self-stabilizing maleimide is a maleimide group describedin Lyon, et al., “Self-hydrolyzing maleimides improve the stability andpharmacological properties of antibody-drug conjugates,” Nat.Biotechnol. 32(10):1059-1062 (2014). In some instances, the linkercomprises a self-stabilizing maleimide. In some instances, the linker isa self-stabilizing maleimide.

In some embodiments, the linker comprises a peptide moiety. In someinstances, the peptide moiety comprises at least 2, 3, 4, 5, or 6 moreamino acid residues. In some instances, the peptide moiety comprises atmost 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In some instances, thepeptide moiety comprises about 2, about 3, about 4, about 5, or about 6amino acid residues. In some instances, the peptide moiety is acleavable peptide moiety (e.g., either enzymatically or chemically). Insome instances, the peptide moiety is a non-cleavable peptide moiety. Insome instances, the peptide moiety comprises Val-Cit(valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 14223), Phe-Lys,Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg,Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 14224),or Gly-Phe-Leu-Gly (SEQ ID NO: 14225). In some instances, the linkercomprises a peptide moiety such as: Val-Cit (valine-citrulline),Gly-Gly-Phe-Gly (SEQ ID NO: 14223), Phe-Lys, Val-Lys, Gly-Phe-Lys,Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit,Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 14224), or Gly-Phe-Leu-Gly(SEQ ID NO: 14225). In some cases, the linker comprises Val-Cit. In somecases, the linker is Val-Cit.

In some embodiments, the linker comprises a benzoic acid group, or itsderivatives thereof. In some instances, the benzoic acid group or itsderivatives thereof comprise paraaminobenzoic acid (PABA). In someinstances, the benzoic acid group or its derivatives thereof comprisegamma-aminobutyric acid (GABA).

In some embodiments, the linker comprises one or more of a maleimidegroup, a peptide moiety, and/or a benzoic acid group, in anycombination. In some embodiments, the linker comprises a combination ofa maleimide group, a peptide moiety, and/or a benzoic acid group. Insome instances, the maleimide group is maleimidocaproyl (me). In someinstances, the peptide group is val-cit. In some instances, the benzoicacid group is PABA. In some instances, the linker comprises a me-val-citgroup. In some cases, the linker comprises a val-cit-PABA group. Inadditional cases, the linker comprises a mc-val-cit-PABA group.

In some embodiments, the linker is a self-immolative linker or aself-elimination linker. In some cases, the linker is a self-immolativelinker. In other cases, the linker is a self-elimination linker (e.g., acyclization self-elimination linker). In some instances, the linkercomprises a linker described in U.S. Pat. No. 9,089,614 or PCTPublication No. WO2015038426.

In some embodiments, the linker is a dendritic type linker. In someinstances, the dendritic type linker comprises a branching,multifunctional linker moiety. In some instances, the dendritic typelinker is used to increase the molar ratio of polynucleotide B to thebinding moiety A. In some instances, the dendritic type linker comprisesPAMAM dendrimers.

In some embodiments, the linker is a traceless linker or a linker inwhich after cleavage does not leave behind a linker moiety (e.g., anatom or a linker group) to a binding moiety A, a polynucleotide B, apolymer C, or an endosomolytic moiety D. Exemplary traceless linkersinclude, but are not limited to, germanium linkers, silicium linkers,sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers,boron linkers, chromium linkers, or phenylhydrazide linker. In somecases, the linker is a traceless aryl-triazene linker as described inHejesen, et al., “A traceless aryl-triazene linker for DNA-directedchemistry,” Org Biomol Chem 11(15): 2493-2497 (2013). In some instances,the linker is a traceless linker described in Blaney, et al., “Tracelesssolid-phase organic synthesis,” Chem. Rev. 102: 2607-2024 (2002). Insome instances, a linker is a traceless linker as described in U.S. Pat.No. 6,821,783.

In some instances, the linker is a linker described in U.S. Pat. Nos.6,884,869; 7,498,298; 8,288,352; 8,609,105; or 8,697,688; U.S. PatentPublication Nos. 2014/0127239; 2013/028919; 2014/286970; 2013/0309256;2015/037360; or 2014/0294851; or PCT Publication Nos. WO2015057699;WO2014080251; WO2014197854; WO2014145090; or WO2014177042.

In some embodiments, X₁ and X₂ are each independently a bond or anon-polymeric linker. In some instances, X₁ and X₂ are eachindependently a bond. In some cases, X₁ and X₂ are each independently anon-polymeric linker.

In some instances, X₁ is a bond or a non-polymeric linker. In someinstances, X₁ is a bond. In some instances, X₁ is a non-polymericlinker. In some instances, the linker is a C₁-C₆ alkyl group. In somecases, X₁ is a C₁-C₆ alkyl group, such as for example, a C₅, C₄, C₃, C₂,or C₁ alkyl group. In some cases, the C₁-C₆ alkyl group is anunsubstituted C₁-C₆ alkyl group. As used in the context of a linker, andin particular in the context of X₁, alkyl means a saturated straight orbranched hydrocarbon radical containing up to six carbon atoms. In someinstances, X₁ includes a homobifunctional linker or a heterobifunctionallinker described supra. In some cases, X₁ includes a heterobifunctionallinker. In some cases, X₁ includes sMCC. In other instances, X₁ includesa heterobifunctional linker optionally conjugated to a C₁-C₆ alkylgroup. In other instances, X₁ includes sMCC optionally conjugated to aC₁-C₆ alkyl group. In additional instances, X₁ does not include ahomobifunctional linker or a heterobifunctional linker described supra.

In some instances, X₂ is a bond or a linker. In some instances, X₂ is abond. In other cases, X₂ is a linker. In additional cases, X₂ is anon-polymeric linker. In some embodiments, X₂ is a C₁-C₆ alkyl group. Insome instances, X₂ is a homobifunctional linker or a heterobifunctionallinker described supra. In some instances, X₂ is a homobifunctionallinker described supra. In some instances, X₂ is a heterobifunctionallinker described supra. In some instances, X₂ comprises a maleimidegroup, such as maleimidocaproyl (me) or a self-stabilizing maleimidegroup described above. In some instances, X₂ comprises a peptide moiety,such as Val-Cit. In some instances, X₂ comprises a benzoic acid group,such as PABA. In additional instances, X₂ comprises a combination of amaleimide group, a peptide moiety, and/or a benzoic acid group. Inadditional instances, X₂ comprises a me group. In additional instances,X₂ comprises a me-val-cit group. In additional instances, X₂ comprises aval-cit-PABA group. In additional instances, X₂ comprises ame-val-cit-PABA group.

Methods of Use

Muscle atrophy refers to a loss of muscle mass and/or to a progressiveweakening and degeneration of muscles. In some cases, the loss of musclemass and/or the progressive weakening and degeneration of muscles occursdue to a high rate of protein degradation, a low rate of proteinsynthesis, or a combination of both. In some cases, a high rate ofmuscle protein degradation is due to muscle protein catabolism (i.e.,the breakdown of muscle protein in order to use amino acids assubstrates for gluconeogenesis).

In one embodiment, muscle atrophy refers to a significant loss in musclestrength. By significant loss in muscle strength is meant a reduction ofstrength in diseased, injured, or unused muscle tissue in a subjectrelative to the same muscle tissue in a control subject. In anembodiment, a significant loss in muscle strength is a reduction instrength of at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, ormore relative to the same muscle tissue in a control subject. In anotherembodiment, by significant loss in muscle strength is meant a reductionof strength in unused muscle tissue relative to the muscle strength ofthe same muscle tissue in the same subject prior to a period of nonuse.In an embodiment, a significant loss in muscle strength is a reductionof at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,at least 35%, at least 40%, at least 45%, at least 50%, or more relativeto the muscle strength of the same muscle tissue in the same subjectprior to a period of nonuse.

In another embodiment, muscle atrophy refers to a significant loss inmuscle mass. By significant loss in muscle mass is meant a reduction ofmuscle volume in diseased, injured, or unused muscle tissue in a subjectrelative to the same muscle tissue in a control subject. In anembodiment, a significant loss of muscle volume is at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, or more relative to the samemuscle tissue in a control subject. In another embodiment, bysignificant loss in muscle mass is meant a reduction of muscle volume inunused muscle tissue relative to the muscle volume of the same muscletissue in the same subject prior to a period of nonuse. In anembodiment, a significant loss in muscle tissue is at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, or more relative to the musclevolume of the same muscle tissue in the same subject prior to a periodof nonuse. Muscle volume is optionally measured by evaluating thecross-section area of a muscle such as by Magnetic Resonance Imaging(e.g., by a muscle volume/cross-section area (CSA) MRI method).

Myotonic dystrophy is a multisystemic neuromuscular disease comprisingtwo main types: myotonic dystrophy type 1 (DM1) and myotonic dystrophytype 2 (DM2). DM1 is caused by a dominantly inherited “CTG” repeatexpansion in the gene DM protein kinase (DMPK), which when transcribedinto mRNA, forms hairpins that bind with high affinity to theMuscleblind-like (MBNL) family of proteins. MBNL proteins are involvedin post-transcriptional splicing and polyadenylatin site regulation andloss of the MBNL protein functions lead to downstream accumulation ofnuclear foci and increase in mis-splicing events and subsequently tomyotonia and other clinical symptoms.

In some embodiments, described herein is a method of treating muscleatrophy or myotonic dystrophy in a subject, which comprisesadministering to the subject a therapeutically effective amount of apolynucleic acid molecule described herein or a polynucleic acidmolecule conjugate described herein. In some instances, the muscleatrophy is associated and/or induced by cachexia (e.g., cancercachexia), denervation, myopathy, motor neuron diseases, diabetes,chronic obstructive pulmonary disease, liver disease, congestive heartfailure, chronic renal failure, chronic infection, sepsis, fasting,sarcopenia, glucocorticoid-induced atrophy, disuse, or space flight. Insome cases, myotonic dystrophy is DM1.

Cachexia

Cachexia is an acquired, accelerated loss of muscle caused by anunderlying disease. In some instances, cachexia refers to a loss of bodymass that cannot be reversed nutritionally, and is generally associatedwith an underlying disease, such as cancer, COPD, AIDS, heart failure,and the like. When cachexia is seen in a patient with end-stage cancer,it is called “cancer cachexia”. Cancer cachexia affects the majority ofpatients with advanced cancer and is associated with a reduction intreatment tolerance, response to therapy, quality of life and durationof survival. It some instances, cancer cachexia is defined as amultifactorial syndrome characterized by an ongoing loss of skeletalmuscle mass, with or without loss of fat mass, which cannot be fullyreversed by conventional nutritional support and leads to progressivefunctional impairment. In some cases, skeletal muscle loss appears to bethe most significant event in cancer cachexia. In addition, theclassification of cancer cachexia suggests that the diagnostic criteriatakes into account not only that weight loss is a signal event of thecachectic process but that the initial reserve of the patient shouldalso be considered, such as low BMI or low level of muscularity.

In some embodiments, described herein is a method of treatingcachexia-associated muscle atrophy in a subject, which comprisesadministering to the subject a therapeutically effective amount of apolynucleic acid molecule described herein or a polynucleic acidmolecule conjugate described herein. In additional embodiments,described herein is a method of treating cancer cachexia-associatedmuscle atrophy in a subject, which comprises administering to thesubject a therapeutically effective amount of a polynucleic acidmolecule described herein or a polynucleic acid molecule conjugatedescribed herein.

Denervation

Denervation is an injury to the peripheral motoneurons with a partial orcomplete interruption of the nerve fibers between an organ and thecentral nervous system, resulting in an interruption of nerve conductionand motoneuron firing which, in turn, prevents the contractability ofskeletal muscles. This loss of nerve function is either localized orgeneralized due to the loss of an entire motor neuron unit. Theresulting inability of skeletal muscles to contract leads to muscleatrophy. In some instances, denervation is associated with or as aresult of degenerative, metabolic, or inflammatory neuropathy (e.g.,Guillain-Barre syndrome, peripheral neuropathy, or exposure toenvironmental toxins or drugs). In additional instances, denervation isassociated with a physical injury, e.g., a surgical procedure.

In some embodiments, described herein is a method of treating muscleatrophy associated with or induced by denervation in a subject, whichcomprises administering to the subject a therapeutically effectiveamount of a polynucleic acid molecule described herein. In otherembodiments, described herein is a method of treating muscle atrophyassociated with or induced by denervation in a subject, which comprisesadministering to the subject a therapeutically effective amount of apolynucleic acid molecule conjugate described herein.

Myopathy

Myopathy is an umbrella term that describes a disease of the muscle. Insome instances, myopathy includes myotonia; congenital myopathy such asnemaline myopathy, multi/minicore myopathy and myotubular(centronuclear) myopathy; mitochondrial myopathy; familial periodicparalysis; inflammatory myopathy; metabolic myopathy, for example,caused by a glycogen or lipid storage disease; dermatomyositis;polymyositis; inclusion body myositis; myositis ossificans;rhabdomyolysis; and myoglobinurias. In some instances, myopathy iscaused by a muscular dystrophy syndrome, such as Duchenne, Becker,myotonic, fascioscapulohumeral, Emery-Dreifuss, oculopharyngeal,scapulohumeral, limb girdle, Fukuyama, a congenital muscular dystrophy,or hereditary distal myopathy. In some instances, myopathy is caused bymyotonic dystrophy (e.g., myotonic dystrophy type 1 or DM1). In someinstances, myopathy is caused by DM1.

In some embodiments, described herein is a method of treating muscleatrophy associated with or induced by myopathy in a subject, whichcomprises administering to the subject a therapeutically effectiveamount of a polynucleic acid molecule described herein. In otherembodiments, described herein is a method of treating muscle atrophyassociated with or induced by myopathy in a subject, which comprisesadministering to the subject a therapeutically effective amount of apolynucleic acid molecule conjugate described herein.

Motor Neuron Diseases

Motor neuron disease (MND) encompasses a neurological disorder thataffects motor neurons, cells that control voluntary muscles of the body.Exemplary motor neuron diseases include, but are not limited to, adultmotor neuron diseases, infantile spinal muscular atrophy, amyotrophiclateral sclerosis, juvenile spinal muscular atrophy, autoimmune motorneuropathy with multifocal conductor block, paralysis due to stroke orspinal cord injury, or skeletal immobilization due to trauma.

In some embodiments, described herein is a method of treating muscleatrophy associated with or induced by a motor neuron disease in asubject, which comprises administering to the subject a therapeuticallyeffective amount of a polynucleic acid molecule described herein. Inother embodiments, described herein is a method of treating muscleatrophy associated with or induced by a motor neuron disease in asubject, which comprises administering to the subject a therapeuticallyeffective amount of a polynucleic acid molecule conjugate describedherein.

Diabetes

Diabetes (diabetes mellitus, DM) comprises type 1 diabetes, type 2diabetes, type 3 diabetes, type 4 diabetes, double diabetes, latentautoimmune diabetes (LAD), gestational diabetes, neonatal diabetesmellitus (NDM), maturity onset diabetes of the young (MODY), Wolframsyndrome, Alström syndrome, prediabetes, or diabetes insipidus. Type 2diabetes, also called non-insulin dependent diabetes, is the most commontype of diabetes accounting for 95% of all diabetes cases. In someinstances, type 2 diabetes is caused by a combination of factors,including insulin resistance due to pancreatic beta cell dysfunction,which in turn leads to high blood glucose levels. In some cases,increased glucagon levels stimulate the liver to produce an abnormalamount of unneeded glucose, which contributes to high blood glucoselevels.

Type 1 diabetes, also called insulin-dependent diabetes, comprises about5% to 10% of all diabetes cases. Type 1 diabetes is an autoimmunedisease where T cells attack and destroy insulin-producing beta cells inthe pancreas. In some embodiments, Type 1 diabetes is caused by geneticand environmental factors.

Type 4 diabetes is a recently discovered type of diabetes affectingabout 20% of diabetic patients age 65 and over. In some embodiments,type 4 diabetes is characterized by age-associated insulin resistance.

In some embodiments, type 3 diabetes is used as a term for Alzheimer'sdisease resulting in insulin resistance in the brain.

In some embodiments, described herein is a method of treatingdiabetes-associated muscle atrophy in a subject, which comprisesadministering to the subject a therapeutically effective amount of apolynucleic acid molecule described herein or a polynucleic acidmolecule conjugate described herein. In additional embodiments,described herein is a method of treating cancer diabetes-associatedmuscle atrophy in a subject, which comprises administering to thesubject a therapeutically effective amount of a polynucleic acidmolecule described herein or a polynucleic acid molecule conjugatedescribed herein.

Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a type of obstructivelung disease characterized by long-term breathing problems and poorairflow. Chronic bronchitis and emphysema are two different types ofCOPD. In some instances, described herein is a method of treating muscleatrophy associated with or induced by COPD (e.g., chronic bronchitis oremphysema) in a subject, which comprises administering to the subject atherapeutically effective amount of a polynucleic acid moleculedescribed herein. In other embodiments, described herein is a method oftreating muscle atrophy associated with or induced by COPD (e.g.,chronic bronchitis or emphysema) in a subject, which comprisesadministering to the subject a therapeutically effective amount of apolynucleic acid molecule conjugate described herein.

Liver Diseases

Liver disease (or hepatic disease) comprises fibrosis, cirrhosis,hepatitis, alcoholic liver disease, hepatic steatosis, a hereditarydisease, or primary liver cancer. In some instances, described herein isa method of treating muscle atrophy associated with or induced by aliver disease in a subject, which comprises administering to the subjecta therapeutically effective amount of a polynucleic acid moleculedescribed herein. In other embodiments, described herein is a method oftreating muscle atrophy associated with or induced by a liver disease ina subject, which comprises administering to the subject atherapeutically effective amount of a polynucleic acid moleculeconjugate described herein.

Congestive Heart Failure

Congestive heart failure is a condition in which the heart is unable topump enough blood and oxygen to the body's tissues. In some instances,described herein is a method of treating muscle atrophy associated withor induced by congestive heart failure in a subject, which comprisesadministering to the subject a therapeutically effective amount of apolynucleic acid molecule described herein. In other embodiments,described herein is a method of treating muscle atrophy associated withor induced by congestive heart failure in a subject, which comprisesadministering to the subject a therapeutically effective amount of apolynucleic acid molecule conjugate described herein.

Chronic Renal Failure

Chronic renal failure or chronic kidney disease is a conditioncharacterized by a gradual loss of kidney function over time. In someinstances, described herein is a method of treating muscle atrophyassociated with or induced by a chronic renal failure in a subject,which comprises administering to the subject a therapeutically effectiveamount of a polynucleic acid molecule described herein. In otherembodiments, described herein is a method of treating muscle atrophyassociated with or induced by a chronic renal failure in a subject,which comprises administering to the subject a therapeutically effectiveamount of a polynucleic acid molecule conjugate described herein.

Chronic Infections

In some embodiments, chronic infection such as AIDS further leads tomuscle atrophy. In some instances, described herein is a method oftreating muscle atrophy associated with or induced by a chronicinfection (e.g., AIDS) in a subject, which comprises administering tothe subject a therapeutically effective amount of a polynucleic acidmolecule described herein. In other embodiments, described herein is amethod of treating muscle atrophy associated with or induced by achronic infection (e.g., AIDS) in a subject, which comprisesadministering to the subject a therapeutically effective amount of apolynucleic acid molecule conjugate described herein.

Sepsis

Sepsis is an immune response to an infection leading to tissue damage,organ failure, and/or death. In some embodiments, described herein is amethod of treating muscle atrophy associated with or induced by sepsisin a subject, which comprises administering to the subject atherapeutically effective amount of a polynucleic acid moleculedescribed herein. In other embodiments, described herein is a method oftreating muscle atrophy associated with or induced by sepsis in asubject, which comprises administering to the subject a therapeuticallyeffective amount of a polynucleic acid molecule conjugate describedherein.

Fasting

Fasting is a willing abstinence or reduction from some or all food,drinks, or both, for a period of time. In some embodiments, describedherein is a method of treating muscle atrophy associated with or inducedby fasting in a subject, which comprises administering to the subject atherapeutically effective amount of a polynucleic acid moleculedescribed herein. In other embodiments, described herein is a method oftreating muscle atrophy associated with or induced by fasting in asubject, which comprises administering to the subject a therapeuticallyeffective amount of a polynucleic acid molecule conjugate describedherein.

Sarcopenia

Sarcopenia is the continuous process of muscle atrophy in the course ofregular aging that is characterized by a gradual loss of muscle mass andmuscle strength over a span of months and years. A regular aging processmeans herein an aging process that is not influenced or accelerated bythe presence of disorders and diseases which promote skeletomuscularneurodegeneration.

In some embodiments, described herein is a method of treating muscleatrophy associated with or induced by sarcopenia in a subject, whichcomprises administering to the subject a therapeutically effectiveamount of a polynucleic acid molecule described herein. In otherembodiments, described herein is a method of treating muscle atrophyassociated with or induced by sarcopenia in a subject, which comprisesadministering to the subject a therapeutically effective amount of apolynucleic acid molecule conjugate described herein.

Glucocorticoid-Associated Muscle Atrophy

In some embodiments, treatment with a glucocorticoid further results inmuscle atrophy. Exemplary glucocorticoids include, but are not limitedto, cortisol, dexamethasone, betamethasone, prednisone,methylprednisolone, and prednisolone.

In some embodiments, described herein is a method of treatingglucocorticoid-associated muscle atrophy in a subject, which comprisesadministering to the subject a therapeutically effective amount of apolynucleic acid molecule described herein. In other embodiments,described herein is a method of treating glucocorticoid-associatedmuscle atrophy in a subject, which comprises administering to thesubject a therapeutically effective amount of a polynucleic acidmolecule conjugate described herein.

Disuse-Associated Muscle Atrophy

Disuse-associated muscle atrophy results when a limb is immobilized(e.g., due to a limb or joint fracture or an orthopedic surgery such asa hip or knee replacement surgery). As used herein, “immobilization” or“immobilized” refers to the partial or complete restriction of movementof limbs, muscles, bones, tendons, joints, or any other body parts foran extended period of time (e.g., for 2 days, 3 days, 4 days, 5 days, 6days, a week, two weeks, or more). In some instances, a period ofimmobilization includes short periods or instances of unrestrainedmovement, such as to bathe, to replace an external device, or to adjustan external device. Limb immobilization is optionally carried out by anyvariety of external devices including, but are not limited to, braces,slings, casts, bandages, and splints (any of which is optionallycomposed of hard or soft material including but not limited to cloth,gauze, fiberglass, plastic, plaster, or metal), as well as any varietyof internal devices including surgically implanted splints, plates,braces, and the like. In the context of limb immobilization, therestriction of movement involves a single joint or multiple joints(e.g., simple joints such as the shoulder joint or hip joint, compoundjoints such as the radiocarpal joint, and complex joints such as theknee joint, including but not limited to one or more of the following:articulations of the hand, shoulder joints, elbow joints, wrist joints,auxiliary articulations, sternoclavicular joints, vertebralarticulations, temporomandibular joints, sacroiliac joints, hip joints,knee joints, and articulations of the foot), a single tendon or ligamentor multiple tendons or ligaments (e.g., including but not limited to oneor more of the following: the anterior cruciate ligament, the posteriorcruciate ligament, rotator cuff tendons, medial collateral ligaments ofthe elbow and knee, flexor tendons of the hand, lateral ligaments of theankle, and tendons and ligaments of the jaw or temporomandibular joint),a single bone or multiple bones (e.g., including but not limited to oneor more of the Wowing: the skull, mandible, clavicle, ribs, radius,ulna, humorous, pelvis, sacrum, femur, patella, phalanges, carpals,metacarpals, tarsals, metatarsals, fibula, tibia, scapula, andvertebrae), a single muscle or multiple muscles (e.g., including but notlimited to one or more of the following: latissimus dorsi, trapezius,deltoid, pectorals, biceps, triceps, external obliques, abdominals,gluteus maximus, hamstrings, quadriceps, gastrocnemius, and diaphragm);a single limb or multiple limbs one or more of the arms and legs), orthe entire skeletal muscle system or portions thereof (e.g., in the caseof a full body cast or spica cast).

In some embodiments, described herein is a method of treatingdisuse-associated muscle atrophy in a subject, which comprisesadministering to the subject a therapeutically effective amount of apolynucleic acid molecule described herein. In other embodiments,described herein is a method of treating disuse-associated muscleatrophy in a subject, which comprises administering to the subject atherapeutically effective amount of a polynucleic acid moleculeconjugate described herein.

Pharmaceutical Formulation

In some embodiments, the pharmaceutical formulations described hereinare administered to a subject by multiple administration routes,including but not limited to, parenteral (e.g., intravenous,subcutaneous, intramuscular), oral, intranasal, buccal, rectal, ortransdermal administration routes. In some instances, the pharmaceuticalcomposition describe herein is formulated for parenteral (e.g.,intravenous, subcutaneous, intramuscular, intra-arterial,intraperitoneal, intrathecal, intracerebral, intracerebroventricular, orintracranial) administration. In other instances, the pharmaceuticalcomposition describe herein is formulated for oral administration. Instill other instances, the pharmaceutical composition describe herein isformulated for intranasal administration.

In some embodiments, the pharmaceutical formulations include, but arenot limited to, aqueous liquid dispersions, self-emulsifyingdispersions, solid solutions, liposomal dispersions, aerosols, soliddosage forms, powders, immediate release formulations, controlledrelease formulations, fast melt formulations, tablets, capsules, pills,delayed release formulations, extended release formulations, pulsatilerelease formulations, multiparticulate formulations (e.g., nanoparticleformulations), and mixed immediate and controlled release formulations.

In some instances, the pharmaceutical formulation includesmultiparticulate formulations. In some instances, the pharmaceuticalformulation includes nanoparticle formulations. In some instances,nanoparticles comprise cMAP, cyclodextrin, or lipids. In some cases,nanoparticles comprise solid lipid nanoparticles, polymericnanoparticles, self-emulsifying nanoparticles, liposomes,microemulsions, or micellar solutions. Additional exemplarynanoparticles include, but are not limited to, paramagneticnanoparticles, superparamagnetic nanoparticles, metal nanoparticles,fullerene-like materials, inorganic nanotubes, dendrimers (such as withcovalently attached metal chelates), nanofibers, nanohorns, nano-onions,nanorods, nanoropes and quantum dots. In some instances, a nanoparticleis a metal nanoparticle, e.g., a nanoparticle of scandium, titanium,vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc,yttrium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium,silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium,platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium,lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium,potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, andcombinations, alloys or oxides thereof.

In some instances, a nanoparticle includes a core or a core and a shell,as in a core-shell nanoparticle.

In some instances, a nanoparticle is further coated with molecules forattachment of functional elements (e.g., with one or more of apolynucleic acid molecule or binding moiety described herein). In someinstances, a coating comprises chondroitin sulfate, dextran sulfate,carboxymethyl dextran, alginic acid, pectin, carragheenan, fucoidan,agaropectin, porphyran, karaya gum, gellan gum, xanthan gum, hyaluronicacids, glucosamine, galactosamine, chitin (or chitosan), polyglutamicacid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease,trypsinogen, chymotrypsinogen, α-chymotrypsin, polylysine, polyarginine,histone, protamine, ovalbumin or dextrin or cyclodextrin. In someinstances, a nanoparticle comprises a graphene-coated nanoparticle.

In some cases, a nanoparticle has at least one dimension of less thanabout 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm.

In some instances, the nanoparticle formulation comprises paramagneticnanoparticles, superparamagnetic nanoparticles, metal nanoparticles,fullerene-like materials, inorganic nanotubes, dendrimers (such as withcovalently attached metal chelates), nanofibers, nanohorns, nano-onions,nanorods, nanoropes or quantum dots. In some instances, a polynucleicacid molecule or a binding moiety described herein is conjugated eitherdirectly or indirectly to the nanoparticle. In some instances, at least1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more polynucleicacid molecules or binding moieties described herein are conjugatedeither directly or indirectly to a nanoparticle.

In some embodiments, the pharmaceutical formulation comprises a deliveryvector, e.g., a recombinant vector, the delivery of the polynucleic acidmolecule into cells. In some instances, the recombinant vector is DNAplasmid. In other instances, the recombinant vector is a viral vector.Exemplary viral vectors include vectors derived from adeno-associatedvirus, retrovirus, adenovirus, or alphavirus. In some instances, therecombinant vectors capable of expressing the polynucleic acid moleculesprovide stable expression in target cells. In additional instances,viral vectors are used that provide for transient expression ofpolynucleic acid molecules.

In some embodiments, the pharmaceutical formulation includes a carrieror carrier materials selected on the basis of compatibility with thecomposition disclosed herein, and the release profile properties of thedesired dosage form. Exemplary carrier materials include, e.g., binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, diluents, and thelike. Pharmaceutically compatible carrier materials include, but are notlimited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerine, magnesiumsilicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters,sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine,sodium chloride, tricalcium phosphate, dipotassium phosphate, celluloseand cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like. See,e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed(Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999).

In some instances, the pharmaceutical formulation further includes pHadjusting agents or buffering agents which include acids such as acetic,boric, citric, lactic, phosphoric and hydrochloric acids; bases such assodium hydroxide, sodium phosphate, sodium borate, sodium citrate,sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; andbuffers such as citrate/dextrose, sodium bicarbonate and ammoniumchloride. Such acids, bases and buffers are included in an amountrequired to maintain pH of the composition in an acceptable range.

In some instances, the pharmaceutical formulation includes one or moresalts in an amount required to bring osmolality of the composition intoan acceptable range. Such salts include those having sodium, potassiumor ammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

In some instances, the pharmaceutical formulation further includesdiluent which are used to stabilize compounds because they provide amore stable environment. Salts dissolved in buffered solutions (whichalso provide pH control or maintenance) are utilized as diluents in theart, including, but not limited to a phosphate buffered saline solution.In certain instances, diluents increase bulk of the composition tofacilitate compression or create sufficient bulk for homogenous blendfor capsule filling. Such compounds include e.g., lactose, starch,mannitol, sorbitol, dextrose, microcrystalline cellulose such asAvicel®; dibasic calcium phosphate, dicalcium phosphate dihydrate;tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-driedlactose; pregelatinized starch, compressible sugar, such as Di-Pac®(Amstar); mannitol, hydroxypropylmethylcellulose,hydroxypropylnethylcellulose acetate stearate, sucrose-based diluents,confectioner's sugar; monobasic calcium sulfate monohydrate, calciumsulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzedcereal solids, amylose; powdered cellulose, calcium carbonate; glycine,kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

In some cases, the pharmaceutical formulation includes disintegrationagents or disintegrants to facilitate the breakup or disintegration of asubstance. The term “disintegrate” include both the dissolution anddispersion of the dosage form when contacted with gastrointestinalfluid. Examples of disintegration agents include a starch, e.g., anatural starch such as corn starch or potato starch, a pregelatinizedstarch such as National 1551 or Amijel®, or sodium starch glycolate suchas Promogel® or Explotab®, a cellulose such as a wood product,methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel®PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, andSolka-Floc®, methylcellulose, croscarmellose, or a cross-linkedcellulose, such as cross-linked sodium carboxymethylcellulose(Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linkedcroscarmellose, a cross-linked starch such as sodium starch glycolate, across-linked polymer such as crospovidone, a cross-linkedpolyvinylpyrrolidone, alginate such as alginic acid or a salt of alginicacid such as sodium alginate, a clay such as Veegum® HV (magnesiumaluminum silicate), a gum such as agar, guar, locust bean, Karaya,pectin, or tragacanth, sodium starch glycolate, bentonite, a naturalsponge, a surfactant, a resin such as a cation-exchange resin, citruspulp, sodium lauryl sulfate, sodium lauryl sulfate in combinationstarch, and the like.

In some instances, the pharmaceutical formulation includes fillingagents such as lactose, calcium carbonate, calcium phosphate, dibasiccalcium phosphate, calcium sulfate, microcrystalline cellulose,cellulose powder, dextrose, dextrates, dextran, starches, pregelatinizedstarch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride,polyethylene glycol, and the like.

Lubricants and glidants are also optionally included in thepharmaceutical formulations described herein for preventing, reducing orinhibiting adhesion or friction of materials. Exemplary lubricantsinclude, e.g., stearic acid, calcium hydroxide, talc, sodium stearylfumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetableoil such as hydrogenated soybean oil (Sterotex®), higher fatty acids andtheir alkali-metal and alkaline earth metal salts, such as aluminum,calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol,tale, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate,sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or amethoxypolyethylene glycol such as Carbowax™, sodium oleate, sodiumbenzoate, glyceryl behenate, polyethylene glycol, magnesium or sodiumlauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil®, a starchsuch as corn starch, silicone oil, a surfactant, and the like.

Plasticizers include compounds used to soften the microencapsulationmaterial or film coatings to make them less brittle. Suitableplasticizers include, e.g., polyethylene glycols such as PEG 300, PEG400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propyleneglycol, oleic acid, triethyl cellulose and triacetin. Plasticizers alsofunction as dispersing agents or wetting agents.

Solubilizers include compounds such as triacetin, triethylcitrate, ethyloleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate,vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone,N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropylalcohol, cholesterol, bile salts, polyethylene glycol 200-600,glycofurol, transcutol, propylene glycol, and dimethyl isosorbide andthe like.

Stabilizers include compounds such as any antioxidation agents, buffers,acids, preservatives and the like.

Suspending agents include compounds such as polyvinylpyrrolidone, e.g.,polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetatecopolymer (S630), polyethylene glycol, e.g., the polyethylene glycol hasa molecular weight of about 300 to about 6000, or about 3350 to about4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcelluloseacetate stearate, polysorbate-80, hydroxyethylcellulose, sodiumalginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum,xanthans, including xanthan gum, sugars, cellulosics, such as, e.g.,sodium carboxymethylcellulose, methylcellulose, sodiumcarboxymethylcellulose, hydroxypropylmethylcellulose,hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylatedsorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone andthe like.

Surfactants include compounds such as sodium lauryl sulfate, sodiumdocusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitanmonooleate, polyoxyethylene sorbitan monooleate, polysorbates,polaxomers, bile salts, glyceryl monostearate, copolymers of ethyleneoxide and propylene oxide, e.g., Pluronic® (BASF), and the like.Additional surfactants include polyoxyethylene fatty acid glycerides andvegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40. Sometimes, surfactants is included to enhance physicalstability or for other purposes.

Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum,carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, hydroxypropylmethyl cellulose acetate stearate,hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol,alginates, acacia, chitosans and combinations thereof.

Wetting agents include compounds such as oleic acid, glycerylmonostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamineoleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate,sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium saltsand the like.

Therapeutic Regimens

In some embodiments, the pharmaceutical compositions described hereinare administered for therapeutic applications. In some embodiments, thepharmaceutical composition is administered once per day, twice per day,three times per day or more. The pharmaceutical composition isadministered daily, every day, every alternate day, five days a week,once a week, every other week, two weeks per month, three weeks permonth, once a month, twice a month, three times per month, or more. Thepharmaceutical composition is administered for at least 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, ormore.

In some embodiments, one or more pharmaceutical compositions areadministered simultaneously, sequentially, or at an interval period oftime. In some embodiments, one or more pharmaceutical compositions areadministered simultaneously. In some cases, one or more pharmaceuticalcompositions are administered sequentially. In additional cases, one ormore pharmaceutical compositions are administered at an interval periodof time (e.g., the first administration of a first pharmaceuticalcomposition is on day one followed by an interval of at least 1, 2, 3,4, 5, or more days prior to the administration of at least a secondpharmaceutical composition).

In some embodiments, two or more different pharmaceutical compositionsare coadministered. In some instances, the two or more differentpharmaceutical compositions are coadministered simultaneously. In somecases, the two or more different pharmaceutical compositions arecoadministered sequentially without a gap of time betweenadministrations. In other cases, the two or more differentpharmaceutical compositions are coadministered sequentially with a gapof about 0.5 hour, 1 hour, 2 hour, 3 hour, 12 hours, 1 day, 2 days, ormore between administrations.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the composition is given continuously;alternatively, the dose of the composition being administered istemporarily reduced or temporarily suspended for a certain length oftime (i.e., a “drug holiday”). In some instances, the length of the drugholiday varies between 2 days and 1 year, including by way of exampleonly, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days,15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320days, 350 days, or 365 days. The dose reduction during a drug holiday isfrom 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, can be reduced, as a function ofthe symptoms, to a level at which the improved disease, disorder orcondition is retained.

In some embodiments, the amount of a given agent that correspond to suchan amount varies depending upon factors such as the particular compound,the severity of the disease, the identity (e.g., weight) of the subjector host in need of treatment, but nevertheless is routinely determinedin a manner known in the art according to the particular circumstancessurrounding the case, including, e.g., the specific agent beingadministered, the route of administration, and the subject or host beingtreated. In some instances, the desired dose is conveniently presentedin a single dose or as divided doses administered simultaneously (orover a short period of time) or at appropriate intervals, for example astwo, three, four or more sub-doses per day.

The foregoing ranges are merely suggestive, as the number of variablesin regard to an individual treatment regime is large, and considerableexcursions from these recommended values are not uncommon. Such dosagesis altered depending on a number of variables, not limited to theactivity of the compound used, the disease or condition to be treated,the mode of administration, the requirements of the individual subject,the severity of the disease or condition being treated, and the judgmentof the practitioner.

In some embodiments, toxicity and therapeutic efficacy of suchtherapeutic regimens are determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, including, but notlimited to, the determination of the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between the toxic and therapeuticeffects is the therapeutic index and it is expressed as the ratiobetween LD50 and ED50. Compounds exhibiting high therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesare used in formulating a range of dosage for use in human. The dosageof such compounds lies preferably within a range of circulatingconcentrations that include the ED50 with minimal toxicity. The dosagevaries within this range depending upon the dosage form employed and theroute of administration utilized.

Kits/Article of Manufacture

Disclosed herein, in certain embodiments, are kits and articles ofmanufacture for use with one or more of the compositions and methodsdescribed herein. Such kits include a carrier, package, or containerthat is compartmentalized to receive one or more containers such asvials, tubes, and the like, each of the container(s) comprising one ofthe separate elements to be used in a method described herein. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. In one embodiment, the containers are formed from a variety ofmaterials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, bags, containers, bottles,and any packaging material suitable for a selected formulation andintended mode of administration and treatment.

For example, the container(s) include target nucleic acid moleculedescribed herein. Such kits optionally include an identifyingdescription or label or instructions relating to its use in the methodsdescribed herein.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included.

In one embodiment, a label is on or associated with the container. Inone embodiment, a label is on a container when letters, numbers or othercharacters forming the label are attached, molded or etched into thecontainer itself; a label is associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. In one embodiment, a label is used toindicate that the contents are to be used for a specific therapeuticapplication. The label also indicates directions for use of thecontents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented ina pack or dispenser device which contains one or more unit dosage formscontaining a compound provided herein. The pack, for example, containsmetal or plastic foil, such as a blister pack. In one embodiment, thepack or dispenser device is accompanied by instructions foradministration. In one embodiment, the pack or dispenser is alsoaccompanied with a notice associated with the container in formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the drug for human or veterinary administration.Such notice, for example, is the labeling approved by the U.S. Food andDrug Administration for prescription drugs, or the approved productinsert. In one embodiment, compositions containing a compound providedherein formulated in a compatible pharmaceutical carrier are alsoprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

Certain Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the claimed subject matter belongs. It is to be understoodthat the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictiveof any subject matter claimed. In this application, the use of thesingular includes the plural unless specifically stated otherwise. Itmust be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. In this application, theuse of “or” means “and/or” unless stated otherwise. Furthermore, use ofthe term “including” as well as other forms, such as “include”,“includes,” and “included,” is not limiting.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 μL” means “about 5 μL” and also “5 μL.” Generally, the term“about” includes an amount that would be expected to be withinexperimental error.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

As used herein, the terms “individual(s)”, “subject(s)” and “patient(s)”mean any mammal. In some embodiments, the mammal is a human. In someembodiments, the mammal is a non-human. None of the terms require or arelimited to situations characterized by the supervision (e.g. constant orintermittent) of a health care worker (e.g. a doctor, a registerednurse, a nurse practitioner, a physician's assistant, an orderly or ahospice worker).

The term “therapeutically effective amount” relates to an amount of apolynucleic acid molecule conjugate that is sufficient to provide adesired therapeutic effect in a mammalian subject. In some cases, theamount is single or multiple dose administration to a patient (such as ahuman) for treating, preventing, preventing the onset of, curing,delaying, reducing the severity of, ameliorating at least one symptom ofa disorder or recurring disorder, or prolonging the survival of thepatient beyond that expected in the absence of such treatment.Naturally, dosage levels of the particular polynucleic acid moleculeconjugate employed to provide a therapeutically effective amount vary independence of the type of injury, the age, the weight, the gender, themedical condition of the subject, the severity of the condition, theroute of administration, and the particular inhibitor employed. In someinstances, therapeutically effective amounts of polynucleic acidmolecule conjugate, as described herein, is estimated initially fromcell culture and animal models. For example, IC₅₀ values determined incell culture methods optionally serve as a starting point in animalmodels, while IC₅₀ values determined in animal models are optionallyused to find a therapeutically effective dose in humans.

Skeletal muscle, or voluntary muscle, is generally anchored by tendonsto bone and is generally used to effect skeletal movement such aslocomotion or in maintaining posture. Although some control of skeletalmuscle is generally maintained as an unconscious reflex (e.g., posturalmuscles or the diaphragm), skeletal muscles react to conscious control.Smooth muscle, or involuntary muscle, is found within the walls oforgans and structures such as the esophagus, stomach, intestines,uterus, urethra, and blood vessels.

Skeletal muscle is further divided into two broad types: Type I (or“slow twitch”) and Type II (or “fast twitch”). Type I muscle fibers aredense with capillaries and are rich in mitochondria and myoglobin, whichgives Type I muscle tissue a characteristic red color. In some cases,Type I muscle fibers carries more oxygen and sustain aerobic activityusing fats or carbohydrates for fuel. Type I muscle fibers contract forlong periods of time but with little force. Type II muscle fibers arefurther subdivided into three major subtypes (IIa, IIx, and IIb) thatvary in both contractile speed and force generated. Type II musclefibers contract quickly and powerfully but fatigue very rapidly, andtherefore produce only short, anaerobic bursts of activity before musclecontraction becomes painful.

Unlike skeletal muscle, smooth muscle is not under conscious control.

Cardiac muscle is also an involuntary muscle but more closely resemblesskeletal muscle in structure and is found only in the heart. Cardiac andskeletal muscles are striated in that they contain sarcomeres that arepacked into highly regular arrangements of bundles. By contrast, themyofibrils of smooth muscle cells are not arranged in sarcomeres andtherefore are not striated.

Muscle cells encompass any cells that contribute to muscle tissue.Exemplary muscle cells include myoblasts, satellite cells, myotubes, andmyofibril tissues.

As used here, muscle force is proportional to the cross-sectional area(CSA), and muscle velocity is proportional to muscle fiber length. Thus,comparing the cross-sectional areas and muscle fibers between variouskinds of muscles is capable of providing an indication of muscleatrophy. Various methods are known in the art to measure muscle strengthand muscle weight, see, for example, “Musculoskeletal assessment: Jointrange of motion and manual muscle strength” by Hazel M. Clarkson,published by Lippincott Williams & Wilkins, 2000. The production oftomographic images from selected muscle tissues by computed axialtomography and sonographic evaluation are additional methods ofmeasuring muscle mass.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1. siRNA Sequences and Synthesis

All siRNA single strands were fully assembled on solid phase usingstandard phosphoramidite chemistry and purified over HPLC. Purifiedsingle strands were duplexed to get the double stranded siRNA. All thesiRNA passenger strand contains conjugation handles in differentformats, C6-NH₂ and/or C6-SH, one at each end of the strand. Theconjugation handle or handles were connected to siRNA passenger strandvia inverted abasic phosphodiester or phosphorothioate. Below FIGS.40A-F are representative structures of the formats used in the in vivoexperiments.

Cholesterol-Myostatin siRNA Conjugate

The sequence of the guide/antisense strand was complementary to the genesequence starting a base position 1169 for the mouse mRNA transcript forMSTN (UUAUUAUUUGUUCUUUGCCUU; SEQ ID NO: 14226). Base, sugar andphosphate modifications were used to optimize the potency of the duplexand reduce immunogenicity. All siRNA single strands were fully assembledon solid phase using standard phospharamidite chemistry and purifiedover HPLC. Purified single strands were duplexed to get the doublestranded siRNA. The passenger strand contained a 5′ cholesterol whichwas conjugated as described below in FIG. 1.

Example 2. General Experimental Protocol and Materials

Animals

All animal studies were conducted following protocols in accordance withthe Institutional Animal Care and Use Committee (IACUC) at ExploraBioLabs, which adhere to the regulations outlined in the USDA AnimalWelfare Act as well as the “Guide for the Care and Use of LaboratoryAnimals” (National Research Council publication, 8th Ed., revised in2011). All mice were obtained from either Charles River Laboratories orHarlan Laboratories.

Wild type CD-1 mice (4-6 week old) were dosed via intravenous (iv)injection with the indicated ASCs (or antibody-nucleic acid conjugate)and doses.

Anti-Transferrin Receptor Antibody

Anti-mouse transferrin receptor antibody or CD71 mAb is a rat IgG2asubclass monoclonal antibody that binds mouse CD71 or mouse transferrinreceptor 1 (mTfR1). The antibody was produced by BioXcell and it iscommercially available (Catalog #BE0175).

IgG2a Isotype Control Antibody

Rat IgG2a isotype control antibody was purchased from BioXcell (Clone2A3, Catalog #BE0089) and this antibody is specific to trinitrophenoland does not have any known antigens in mouse.

Anti-EGFR Antibody

Anti-EGFR antibody is a fully human IgG1κ monoclonal antibody directedagainst the human epidermal growth factor receptor (EGFR). It isproduced in the Chinese Hamster Ovary cell line DJT33, which has beenderived from the CHO cell line CHO-K1SV by transfection with a GS vectorcarrying the antibody genes derived from a human anti-EGFR antibodyproducing hybridoma cell line (2F8). Standard mammalian cell culture andpurification technologies are employed in the manufacturing of anti-EGFRantibody.

The theoretical molecular weight (MW) of anti-EGFR antibody withoutglycans is 146.6 kDa. The experimental MW of the major glycosylatedisoform of the antibody is 149 kDa as determined by mass spectrometry.Using SDS-PAGE under reducing conditions the MW of the light chain wasfound to be approximately 25 kDa and the MW of the heavy chain to beapproximately 50 kDa. The heavy chains are connected to each other bytwo inter-chain disulfide bonds, and one light chain is attached to eachheavy chain by a single inter-chain disulfide bond. The light chain hastwo intra-chain disulfide bonds and the heavy chain has four intra-chaindisulfide bonds. The antibody is N-linked glycosylated at Asn305 of theheavy chain with glycans composed of N-acetyl-glucosamine, mannose,fucose and galactose. The predominant glycans present are fucosylatedbi-antennary structures containing zero or one terminal galactoseresidue.

The charged isoform pattern of the IgG1κ antibody has been investigatedusing imaged capillary IEF, agarose IEF and analytical cation exchangeHPLC. Multiple charged isoforms are found, with the main isoform havingan isoelectric point of approximately 8.7.

The major mechanism of action of anti-EGFR antibody is a concentrationdependent inhibition of EGF-induced EGFR phosphorylation in A431 cancercells. Additionally, induction of antibody-dependent cell-mediatedcytotoxicity (ADCC) at low antibody concentrations has been observed inpre-clinical cellular in vitro studies.

In Vitro Evaluation of siRNA Potency and Efficacy

C2C12 myoblasts (ATCC) were grown in DMEM supplemented with 10% v/v FBS.For transfection, cells were plated at a density of 10.000 cells/well in24-well plates, and transfected within 24 hours. C2C12 myotubes weregenerated by incubating confluent C2C12 myoblast cultures in DMEMsupplemented with 2% v/v horse serum for 3-4 days. During and afterdifferentiation the medium was changed daily. Pre-differentiated primaryhuman skeletal muscle cells were obtained from ThermoFisher and platedin DMEM with 2% v/v horse serum according to recommendations by themanufacturer. Human SJCRH30 rhabdomyosarcoma myoblasts (ATCC) were grownin DMEM supplemented with 10% v/v heat-inactivated fetal calf serum, 4.5mg/mL glucose, 4 mM L-glutamine, 10 mM HEPES, and 1 mM sodium pyruvate.For transfections cells were plated in a density of 10.000-20.000cells/well in 24-well plates and transfected within 24 hours. All cellswere transfected with various concentrations of the siRNAs (0.0001-100nM; 10-fold dilutions) using RNAiMax (ThermoFisher) according to therecommendation by the manufacturer. Transfected cells were incubated in5% CO2 at 37° C. for 2 days, then washed with PBS, and harvested in 300ul TRIzol (ThermoFisher) and stored at −80° C. RNA was prepared using aZYMO 96-well RNA kit (ThermoFisher) and relative RNA expression levelsquantified by RT-qPCR using commercially available TaqMan probes(LifeTechnology). Expression data were analyzed using the □□CT methodnormalized to Ppib expression, and are presented as % KD relative tomock-transfected cells. Data were analyzed by nonlinear regression usinga 3 parameter dose response inhibition function (GraphPad Prism 7.02).All knock down results present the maximal observed KD under theseexperimental conditions.

Myostatin ELISA

Myostatin protein in plasma was quantified using the GDF-8 (Myostatin)Quantikine ELISA Immunoassay (part #DGDF80) from R&D Systems accordingto the manufacturer's instructions.

RISC Loading Assay

Specific immunoprecipitation of the RISC from tissue lysates andquantification of small RNAs in the immunoprecipitates were determine bystem-loop PCR, using an adaptation of the assay described by Pei et al.Quantitative evaluation of siRNA delivery in vivo. RNA (2010),16:2553-2563.

Example 3. Conjugate Synthesis

The structures in FIGS. 41A-F illustrate exemplary A-X₁—B—X₂—Y (FormulaI) architectures described herein.

Example 3.1 Antibody siRNA Conjugate Synthesis Using SMCC Linker

FIG. 42 shows Synthesis scheme-1: Antibody-Cys-SMCC-siRNA-PEG conjugatesvia antibody cysteine conjugation.

Step 1: Antibody Interchain Disulfide Reduction with TCEP

Antibody was buffer exchanged with borax buffer (pH 8) and made up to 10mg/ml concentration. To this solution, 2 equivalents of TCEP in waterwas added and rotated for 2 hours at RT. The resultant reaction mixturewas buffer exchanged with pH 7.4 PBS containing 5 mM EDTA and added to asolution of SMCC-C6-siRNA or SMCC-C6-siRNA-C6-NHCO-PEG-XkDa (2equivalents) (X=0.5 kDa to 10 kDa) in pH 7.4 PBS containing 5 mM EDTA atRT and rotated overnight. Analysis of the reaction mixture by analyticalSAX column chromatography showed antibody siRNA conjugate along withunreacted antibody and siRNA.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1 as described in Example 3.4.Fractions containing DAR1 and DAR>2 antibody-siRNA-PEG conjugates wereseparated, concentrated and buffer exchanged with pH 7.4 PBS.

Step-3: Analysis of the Purified Conjugate

The isolated conjugates were characterized by SEC, SAX chromatographyand SDS-PAGE. The purity of the conjugate was assessed by analyticalHPLC using either anion exchange chromatography method-2 or anionexchange chromatography method-3. Both methods are described in Example3.4. Isolated DAR1 conjugates are typically eluted at 9.0±0.3 min onanalytical SAX method and are greater than 90% pure. The typical DAR>2cysteine conjugate contains more than 85% DAR2 and less than 15% DAR3.

FIG. 2 illustrates SAX HPLC chromatogram of TfR mAb-(Cys)-HPRT-PEG5k,DAR1.

FIG. 3 illustrates SEC HPLC chromatogram of TfR mAb-(Cys)-HPRT-PEG5k,DAR1.

Example 3.2. Antibody siRNA Conjugate Synthesis Using Bis-Maleimide(BisMal) Linker

FIG. 43 shows Synthesis scheme-2: Antibody-Cys-BisMal-siRNA-PEGconjugates.

Step 1: Antibody Reduction with TCEP

Antibody was buffer exchanged with borax buffer (pH 8) and made up to 5mg/ml concentration. To this solution, 2 equivalents of TCEP in waterwas added and rotated for 2 hours at RT. The resultant reaction mixturewas exchanged with pH 7.4 PBS containing 5 mM EDTA and added to asolution of BisMal-C6-siRNA-C6-S-NEM (2 equivalents) in pH 7.4 PBScontaining 5 mM EDTA at RT and kept at 4° C. overnight. Analysis of thereaction mixture by analytical SAX column chromatography showed antibodysiRNA conjugate along with unreacted antibody and siRNA.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing DAR1 andDAR2 antibody-siRNA conjugates were separated, concentrated and bufferexchanged with pH 7.4 PBS.

Step-3: Analysis of the Purified Conjugate

The isolated conjugates were characterized by either mass spec orSDS-PAGE. The purity of the conjugate was assessed by analytical HPLCusing either anion exchange chromatography method-2 or 3 as well as sizeexclusion chromatography method-1.

FIG. 4 illustrates an overlay of DAR1 and DAR2 SAX HPLC chromatograms ofTfR1mAb-Cys-BisMal-siRNA conjugates.

FIG. 5 illustrates an overlay of DAR1 and DAR2 SEC HPLC chromatograms ofTfR1mAb-Cys-BisMal-siRNA conjugates.

Example 3.3. Fab′ Generation from mAb and Conjugation to siRNA

FIG. 44 shows Scheme-3: Fab-siRNA conjugate generation.

Step 1: Antibody Digestion with Pepsin

Antibody was buffer exchanged with pH 4.0, 20 mM sodium acetate/aceticacid buffer and made up to 5 mg/ml concentration. Immobilized pepsin(Thermo Scientific, Prod #20343) was added and incubated for 3 hours at37° C. The reaction mixture was filtered using 30 kDa MWCO Amicon spinfilters and pH 7.4 PBS. The retentate was collected and purified usingsize exclusion chromatography to isolate F(ab′)2. The collected F(ab′)2was then reduced by 10 equivalents of TCEP and conjugated withSMCC-C6-siRNA-PEG5 at room temperature in pH 7.4 PBS. Analysis ofreaction mixture on SAX chromatography showed Fab-siRNA conjugate alongwith unreacted Fab and siRNA-PEG.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing DAR1 andDAR2 Fab-siRNA conjugates were separated, concentrated and bufferexchanged with pH 7.4 PBS.

Step-3: Analysis of the Purified Conjugate

The characterization and purity of the isolated conjugate was assessedby analytical HPLC using anion exchange chromatography method-2 or 3 aswell as by SEC method-1.

FIG. 6 illustrates SEC chromatogram of CD71 Fab-Cys-HPRT-PEG5.

FIG. 7 illustrates SAX chromatogram of CD71 Fab-Cys-HPRT-PEG5.

Example 3.4. Purification and Analytical Methods

Anion Exchange Chromatography Method (SAX)-1.

-   -   1. Column: Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5 mm ID×15        cm, 13 um    -   2. Solvent A: 20 mM TRIS buffer, pH 8.0; Solvent B: 20 mM TRIS,        1.5 M NaCl, pH 8.0; Flow Rate: 6.0 ml/min    -   3. Gradient:

a. % A % B Column Volume b. 100 0 1.00 c. 60 40 18.00 d. 40 60 2.00 e.40 60 5.00 f. 0 100 2.00 g. 100 0 2.00

Anion Exchange Chromatography (SAX) Method-2

-   -   1. Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm    -   2. Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80%        10 mM TRIS pH 8, 20% ethanol, 1.5 M NaCl; Flow Rate: 0.75 ml/min    -   3. Gradient

a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d. 11.00 40 60 e. 13.00 40 60f. 15.00 90 10 g. 20.00 90 10

Anion Exchange Chromatography (SAX) Method-3

-   -   1. Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm    -   2. Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80%        10 mM TRIS pH 8, 20% ethanol, 1.5 M NaCl    -   3. Flow Rate: 0.75 ml/min    -   4. Gradient:

a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d. 11.00 40 60 e. 23.00 40 60f. 25.00 90 10 g. 30.00 90 10

Size Exclusion Chromatography (SEC) Method-1

-   -   1. Column: TOSOH Biosciences, TSKgelG3000SW XL, 7.8×300 mm, 5 μM    -   2. Mobile phase: 150 mM phosphate buffer    -   3. Flow Rate: 1.0 ml/min for 15 mins

Example 4. In Vitro Screen: Atrogin-1

Identification of siRNAs for the Regulation of Mouse and Human/NHPAtrogin-1

A bioinformatics screen conducted identified 56 siRNAs (19mers) thatbind specifically to mouse atrogin-1 (Fbxo32; NM_026346.3). In addition,6 siRNAs were identified that target mouse atrogin-1 and human atrogin-1(FBXO32; NM_058229.3). A screen for siRNAs (19mers) targetingspecifically human/NHP atrogin-1 (FBXO32; NM_058229.3) yielded 52candidates (Table 2A-Table 21B). All selected siRNA target sites do notharbor SNPs (pos. 2-18).

Tables 2A and 2B illustrate identified siRNA candidates for theregulation of mouse and human/NHP atrogin-1.

TABLE 2A mouse SEQ x ID exon # human NO: Sequence of total 23mer 19mertarget pos. in site in NM_0263 NM_026 46.3 mouse 346.3 7 1 X GGGCAGCGGC28 CCGGGAUAAA UAC 8 1 X GGCAGCGGCC 29 CGGGAUAAAU ACU 499 3-Feb XAACCAAAACU 30 CAGUACUUCC AUC 553 4-Mar X UACGAAGGAG 31 CGCCAUGGAU ACU590 4 X X GCUUUCAACA 32 GACUGGACUU CUC 631 4 X CAGAAGAUUC 33 AACUACGUAGUAA 694 5 X GAGUGGCAUC 34 GCCCAAAAGA ACU 772 6 X AAGACUUAUA 35CGGGAACUUC UCC 1178 8 X AAGCUUGUAC 36 GAUGUUACCC AAG 1179 8 X AGCUUGUACG37 AUGUUACCCA AGA 1256 9-Aug X X UGGAAGGGCA 38 CUGACCAUCC GUG 1258 9-AugX X GAAGGGCACU 39 GACCAUCCGU GCA 1260 9 X X AGGGCACUGA 40 CCAUCCGUGC ACG1323 9 X AAGACUUUAU 41 CAAUUUGUUC AAG 1401 9 X GGGAGUCGGG 42 ACACUUCAUUUGU 1459 9 X CGGGGGAUAC 43 GUCAUUGAGG AGA 1504 9 X UUGCCGAUGG 44AAAUUUACAA AUG 1880 9 X X CCACACAAUG 45 GUCUACCUCU AAA 1884 9 X XACAAUGGUCU 46 ACCUCUAAAA GCA 2455 9 X CUGAUAGAUG 47 UGUUCGUCUU AAA 25709 X AUCUCAGGGC 48 UUAAGGAGUU AAU 2572 9 X CUCAGGGCUU 49 AAGGAGUUAA UUC2936 9 X CUGAUUUGCA 50 GGGUCUUACA UCU 3006 9 X CUGGUGGCCA 51 AAUUAAGUUGAAU 3007 9 X UGGUGGCCAA 52 AUUAAGUUGA AUU 3115 9 X GAGAUUACAA 53ACAUUGUAAC AGA 3668 9 X CAGCGCAAAA 54 CUAGUUAGCC AGU 3676 9 X AACUAGUUAG55 CCAGUCUUAC AGA 3715 9 X AAGUCAUAUA 56 GCAUCCAUAC ACC 3800 9 XUAGUAGGUGC 57 UUGCAGGUUC UCC 3845 9 X AUGGUAUGUG 58 ACACAACCGA AGA 38569 X CACAACCGAA 59 GAAUCGUUUG ACG 4026 9 X GGCAAGCAAG 60 AUACCCAUAU UAG4095 9 X AGCUCUUAGG 61 ACAUUAAUAG UCU 4139 9 X UGCAGGACUC 62 CCAGACUUAAAAC 4183 9 X CCCAGAACUG 63 CUAGUACAAA AGC 4203 9 X AGCAAGAGGG 64GUGUGGCUAU AGA 4208 9 X GAGGGGUGUG 65 GCUAUAGAAG UUG 4548 9 X GACCAUGUCG66 CUACUACCAU UGC 4554 9 X GUCGCUACUA 67 CCAUUGCUUC AAG 4563 9 XACCAUUGCUU 68 CAAGUGGGUA UCU 4567 9 X UUGCUUCAAG 69 UGGGUAUCUC AGU 46739 X CUGGUUAGUG 70 AUGAUCAACU UCA 4858 9 X UGCCGCUUCA 71 UACGGGAGAA AAA4970 9 X UCGGCUUCAA 72 CGCAUUGUUU AUU 5022 9 X CUGCCUGGUU 73 AUAAAGCAAUAAC 5235 9 X ACCUGUUAGU 74 GCUUAAACAG ACU 5237 9 X CUGUUAGUGC 75UUAAACAGAC UCA 5279 9 X GGGGCAAACG 76 CAGGGGUGUU ACU 5292 9 X GGGUGUUACU77 CUUUGAUAUA UCA 5443 9 X AUCCCAGACU 78 UUAGACCAAA AGG 5640 9 XUUGUGGACGU 79 GUGUAAAUUC AUG 6000 9 X UUCAUUGACC 80 AACCAGUCUU AAG 61059 X UGCCGCAACC 81 UCCCAAGUCA UAU 6530 9 X GAGUAUAGAC 82 AUGCGUGUUA ACU6537 9 X GACAUGCGUG 83 UUAACUAUGC ACA 6608 9 X UUGGUUCCAU 84 CUUUAUACCAAAU 6668 9 X GUGUCUAAGC 85 UUAGAAGCUU UAA 6720 9 X UGGGUUGAAC 86ACUUUAACUA AAC 6797 9 X AUCUGAAUCC 87 UGUAUAACUU AUU 6799 9 X CUGAAUCCUG88 UAUAACUUAU UUG 6803 9 X AUCCUGUAUA 89 ACUUAUUUGC ACA Sequence 19merOf total pos. 23mer in target NM_058 human site in 229.3 NHP NM_058229.3586 x UUCCAGAAGA 90 UUUAACUACG UGG 589 x CAGAAGAUUU 91 AACUACGUGG UCC1068 x AGCGGCAGAU 92 CCGCAAACGA UUA 1071 x GGCAGAUCCG 93 CAAACGAUUA AUU1073 x CAGAUCCGCA 94 AACGAUUAAU UCU 1075 x GAUCCGCAAA 95 CGAUUAAUUC UGU1076 x AUCCGCAAAC 96 GAUUAAUUCU GUC 1077 x UCCGCAAACG 97 AUUAAUUCUG UCA1079 x CGCAAACGAU 98 UAAUUCUGUC AGA 1083 x AACGAUUAAU 99 UCUGUCAGAC AAA1127 x AUGUAUUUCA 100 AACUUGUCCG AUG 1142 x GUCCGAUGUU 101 ACCCAAGGAAAGA 1164 x AGCAGUAUGG 102 AGAUACCCUU CAG 1228 x CCAUCCGUGC 103ACUGCCAAUA ACC 1254 x AGAGCUGCUC 104 CGUUUCACUU UCA 1361 x UGGGAAUAUG105 GCAUUUGGAC ACU 1492 x UGUGAACUUC 106 UCACUAGAAU UGG 1500 xUCUCACUAGA 107 AUUGGUAUGG AAA 1563 x CAGCAAGACU 108 AUAAGGGCAA UAA 1566x CAAGACUAUA 109 AGGGCAAUAA UUC 1635 x UCUUAUAGUU 110 CCCUAGGAAG AAA1679 x AUAGGACGCU 111 UUGUUUACAA UGU 2487 x UUUUCUUUAG 112 GUCCAACAUCAAA 2488 x UUUCUUUAGG 113 UCCAACAUCA AAA 2582 x AGGAGAGGUA 114CCACAAGUUC AUC 2661 x GAGGCAAAUA 115 UCAGCAGGUA ACU 2663 x GGCAAAUAUC116 AGCAGGUAAC UGU 2790 x UUUCCUACAA 117 CAAUGUACAU AUA 2999 xAAGAGACAAG 118 CUAUGAUACA ACA 3875 x GAAAUCAACC 119 UUUAUGGUUC UCU 4036x GUGCCACGUG 120 GUAUCUGUUA AGU 4039 x CCACGUGGUA 121 UCUGUUAAGU AUG4059 x AUGGCCAGAG 122 CCUCACAUAU AAG 4062 x GCCAGAGCCU 123 CACAUAUAAGUGA 4065 x AGAGCCUCAC 124 AUAUAAGUGA AGA 4117 x AUAAUAGUCU 125AUAGAAUUUC UAU 4444 x GCCUAGAGUC 126 UCUUGAGAGU AAA 4653 x GAAAGCAUCC127 CCAAUGUAUC AGU 4665 x AAUGUAUCAG 128 UUGUGAGAUG AUU 4787 xCUACUAGCAC 129 UUGGGCAGUA AGG 5162 x UUAACUAAAC 130 UCUAUCAUCA UUU 5261x CUGGCCUAAA 131 AUCCUAUUAG UGC 5270 x AAUCCUAUUA 132 GUGCUUAAAC AGA5272 x UCCUAUUAGU 133 GCUUAAACAG ACC 5338 x UUUGAUAUAU 134 CUUGGGUCCUUGA 5737 x UGGCUGUUAA 135 CGUUUCCAUU UCA 5739 x GCUGUUAACG 136UUUCCAUUUC AAG 6019 x CUCAGAGGUA 137 CAUUUAAUCC AUC 6059 x CAGGACCAGC138 UAUGAGAUUC AGU 6140 x GGGGGAUUAU 139 UCCAUGAGGC AGC 6431 xGGCUCCAAGC 140 UGUAUUCUAU ACU 6720 x UUUGUACCAG 141 ACGGUGGCAU AUU

TABLE 2B SEQ SEQ exon ID ID # NO: NO: 19mer pos. sense antisense instrand strand NM_026 sequence sequence 346.3 (5′-3′) (5′-3′) 7 1 GCAGC142 AUUUA 256 GGCCC UCCCG GGGAU GGCCG AAAU CUGC 8 1 CAGCG 143 UAUUU 257GCCCG AUCCC GGAUA GGGCC AAUA GCUG 499 3-Feb CCAAA 144 UGGAA 258 ACUCAGUACU GUACU GAGUU UCCA UUGG 553 4-Mar CGAAG 145 UAUCC 259 GAGCG AUGGCCCAUG GCUCC GAUA UUCG 590 4 UUUCA 146 GAAGU 260 ACAGA CCAGU CUGGA CUGUUCUUC GAAA 631 4 GAAGA 147 ACUAC 261 UUCAA GUAGU CUACG UGAAU UAGU CUUC694 5 GUGGC 148 UUCUU 262 AUCGC UUGGG CCAAA CGAUG AGAA CCAC 772 6 GACUU149 AGAAG 263 AUACG UUCCC GGAAC GUAUA UUCU AGUC 1178 8 GCUUG 150 UGGGU264 UACGA AACAU UGUUA CGUAC CCCA AAGC 1179 8 CUUGU 151 UUGGG 265 ACGAUUAACA GUUAC UCGUA CCAA CAAG 1256 9-Aug GAAGG 152 CGGAU 266 GCACU GGUCAGACCA GUGCC UCCG CUUC 1258 9-Aug AGGGC 153 CACGG 267 ACUGA AUGGU CCAUCCAGUG CGUG CCCU 1260 9 GGCAC 154 UGCAC 268 UGACC GGAUG AUCCG GUCAG UGCAUGCC 1323 9 GACUU 155 UGAAC 269 UAUCA AAAUU AUUUG GAUAA UUCA AGUC 1401 9GAGUC 156 AAAUG 270 GGGAC AAGUG ACUUC UCCCG AUUU ACUC 1459 9 GGGGA 157UCCUC 271 UACGU AAUGA CAUUG CGUAU AGGA CCCC 1504 9 GCCGA 158 UUUGU 272UGGAA AAAUU AUUUA UCCAU CAAA CGGC 1880 9 ACACA 159 UAGAG 273 AUGGU GUAGACUACC CCAUU UCUA GUGU 1884 9 AAUGG 160 CUUUU 274 UCUAC AGAGG CUCUA UAGACAAAG CAUU 2455 9 GAUAG 161 UAAGA 275 AUGUG CGAAC UUCGU ACAUC CUUA UAUC2570 9 CUCAG 162 UAACU 276 GGCUU CCUUA AAGGA AGCCC GUUA UGAG 2572 9CAGGG 163 AUUAA 277 CUUAA CUCCU GGAGU UAAGC UAAU CCUG 2936 9 GAUUU 164AUGUA 278 GCAGG AGACC GUCUU CUGCA ACAU AAUC 3006 9 GGUGG 165 UCAAC 279CCAAA UUAAU UUAAG UUGGC UUGA CACC 3007 9 GUGGC 166 UUCAA 280 CAAAU CUUAAUAAGU UUUGG UGAA CCAC 3115 9 GAUUA 167 UGUUA 281 CAAAC CAAUG AUUGU UUUGUAACA AAUC 3668 9 GCGCA 168 UGGCU 282 AAACU AACUA AGUUA GUUUU GCCA GCGC3676 9 CUAGU 169 UGUAA 283 UAGCC GACUG AGUCU GCUAA UACA CUAG 3715 9GUCAU 170 UGUAU 284 AUAGC GGAUG AUCCA CUAUA UACA UGAC 3800 9 GUAGG 171AGAAC 285 UGCUU CUGCA GCAGG AGCAC UUCU CUAC 3845 9 GGUAU 172 UUCGG 286GUGAC UUGUG ACAAC UCACA CGAA UACC 3856 9 CAACC 173 UCAAA 287 GAAGA CGAUUAUCGU CUUCG UUGA GUUG 4026 9 CAAGC 174 AAUAU 288 AAGAU GGGUA ACCCA UCUUGUAUU CUUG 4095 9 CUCUU 175 ACUAU 289 AGGAC UAAUG AUUAA UCCUA UAGU AGAG4139 9 CAGGA 176 UUUAA 290 CUCCC GUCUG AGACU GGAGU UAAA CCUG 4183 9CAGAA 177 UUUUG 291 CUGCU UACUA AGUAC GCAGU AAAA UCUG 4203 9 CAAGA 178UAUAG 292 GGGGU CCACA GUGGC CCCCU UAUA CUUG 4208 9 GGGGU 179 ACUUC 293GUGGC UAUAG UAUAG CCACA AAGU CCCC 4548 9 CCAUG 180 AAUGG 294 UCGCU UAGUAACUAC GCGAC CAUU AUGG 4554 9 CGCUA 181 UGAAG 295 CUACC CAAUG AUUGC GUAGUUUCA AGCG 4563 9 CAUUG 182 AUACC 296 CUUCA CACUU AGUGG GAAGC GUAU AAUG4567 9 GCUUC 183 UGAGA 297 AAGUG UACCC GGUAU ACUUG CUCA AAGC 4673 9GGUUA 184 AAGUU 298 GUGAU GAUCA GAUCA UCACU ACUU AACC 4858 9 CCGCU 185UUUCU 299 UCAUA CCCGU CGGGA AUGAA GAAA GCGG 4970 9 GGCUU 186 UAAAC 300CAACG AAUGC CAUUG GUUGA UUUA AGCC 5022 9 GCCUG 187 UAUUG 301 GUUAU CUUUAAAAGC UAACC AAUA AGGC 5235 9 CUGUU 188 UCUGU 302 AGUGC UUAAG UUAAA CACUACAGA ACAG 5237 9 GUUAG 189 AGUCU 303 UGCUU GUUUA AAACA AGCAC GACU UAAC5279 9 GGCAA 190 UAACA 304 ACGCA CCCCU GGGGU GCGUU GUUA UGCC 5292 9GUGUU 191 AUAUA 305 ACUCU UCAAA UUGAU GAGUA AUAU ACAC 5443 9 CCCAG 192UUUUG 306 ACUUU GUCUA AGACC AAGUC AAAA UGGG 5640 9 GUGGA 193 UGAAU 307CGUGU UUACA GUAAA CACGU UUCA CCAC 6000 9 CAUUG 194 UAAGA 308 ACCAA CUGGUCCAGU UGGUC CUUA AAUG 6105 9 CCGCA 195 AUGAC 309 ACCUC UUGGG CCAAG AGGUUUCAU GCGG 6530 9 GUAUA 1% UUAAC 310 GACAU ACGCA GCGUG UGUCU UUAA AUAC6537 9 CAUGC 197 UGCAU 311 GUGUU AGUUA AACUA ACACG UGCA CAUG 6608 9GGUUC 198 UUGGU 312 CAUCU AUAAA UUAUA GAUGG CCAA AACC 6668 9 GUCUA 199AAAGC 313 AGCUU UUCUA AGAAG AGCUU CUUU AGAC 6720 9 GGUUG 200 UUAGU 314AACAC UAAAG UUUAA UGUUC CUAA AACC 6797 9 CUGAA 201 UAAGU 315 UCCUG UAUACUAUAA AGGAU CUUA UCAG 6799 9 GAAUC 202 AAUAA 316 CUGUA GUUAU UAACU ACAGGUAUU AUUC 6803 9 CCUGU 203 UGCAA 317 AUAAC AUAAG UUAUU UUAUA UGCA CAGG19mer pos. in sense antiense NM_0 strand strand 582 sequence sequence293 (5′-3′) (5′-3′) 586 CCAGA 204 ACGUA 318 AGAUU GUUAA UAACU AUCUU ACGUCUGG 589 GAAGA 205 ACCAC 319 UUUAA GUAGU CUACG UAAAU UGGU CUUC 1068CGGCA 206 AUCGU 320 GAUCC UUGCG GCAAA GAUCU CGAU GCCG 1071 CAGAU 207UUAAU 321 CCGCA CGUUU AACGA GCGGA UUAA UCUG 1073 GAUCC 208 AAUUA 322GCAAA AUCGU CGAUU UUGCG AAUU GAUC 1075 UCCGC 209 AGAAU 323 AAACG UAAUCAUUAA GUUUG UUCU CGGA 1076 CCGCA 210 CAGAA 324 AACGA UUAAU UUAAU CGUUUUCUG GCGG 1077 CGCAA 211 ACAGA 325 ACGAU AUUAA UAAUU UCGUU CUGU UGCG1079 CAAAC 212 UGACA 326 GAUUA GAAUU AUUCU AAUCG GUCA UUUG 1083 CGAUU213 UGUCU 327 AAUUC GACAG UGUCA AAUUA GACA AUCG 1127 GUAUU 214 UCGGA 328UCAAA CAAGU CUUGU UUGAA CCGA AUAC 1142 CCGAU 215 UUUCC 329 GUUAC UUGGGCCAAG UAACA GAAA UCGG 1164 CAGUA 216 GAAGG 330 UGGAG GUAUC AUACC UCCAUCUUC ACUG 1228 AUCCG 217 UUAUU 331 UGCAC GGCAG UGCCA UGCAC AUAA GGAU1254 AGCUG 218 AAAGU 332 CUCCG GAAAC UUUCA GGAGC CUUU AGCU 1361 GGAAU219 UGUCC 333 AUGGC AAAUG AUUUG CCAUA GACA UUCC 1492 UGAAC 220 AAUUC 334UUCUC UAGUG ACUAG AGAAG AAUU UUCA 1500 UCACU 221 UCCAU 335 AGAAU ACCAAUGGUA UUCUA UGGA GUGA 1563 GCAAG 222 AUUGC 336 ACUAU CCUUA AAGGG UAGUCCAAU UUGC 1566 AGACU 223 AUUAU 337 AUAAG UGCCC GGCAA UUAUA UAAU GUCU1635 UUAUA 224 UCUUC 338 GUUCC CUAGG CUAGG GAACU AAGA AUAA 1679 AGGAC225 AUUGU 339 GCUUU AAACA GUUUA AAGCG CAAU UCCU 2487 UUCUU 226 UGAUG 340UAGGU UUGGA CCAAC CCUAA AUCA AGAA 2488 UCUUU 227 UUGAU 341 AGGUC GUUGGCAACA ACCUA UCAA AAGA 2582 GAGAG 228 UGAAC 342 GUACC UUGUG ACAAG GUACCUUCA UCUC 2661 GGCAA 229 UUACC 343 AUAUC UGCUG AGCAG AUAUU GUAA UGCC2663 CAAAU 230 AGUUA 344 AUCAG CCUGC CAGGU UGAUA AACU UUUG 2790 UCCUA231 UAUGU 345 CAACA ACAUU AUGUA GUUGU CAUA AGGA 2999 GAGAC 232 UUGUA 346AAGCU UCAUA AUGAU GCUUG ACAA UCUC 3875 AAUCA 233 AGAAC 347 ACCUU CAUAAUAUGG AGGUU UUCU GAUU 4036 GCCAC 234 UUAAC 348 GUGGU AGAUA AUCUG CCACGUUAA UGGC 4039 ACGUG 235 UACUU 349 GUAUC AACAG UGUUA AUACC AGUA ACGU4059 GGCCA 236 UAUAU 350 GAGCC GUGAG UCACA GCUCU UAUA GGCC 4062 CAGAG237 ACUUA 351 CCUCA UAUGU CAUAU GAGGC AAGU UCUG 4065 AGCCU 238 UUCAC 352CACAU UUAUA AUAAG UGUGA UGAA GGCU 4117 AAUAG 239 AGAAA 353 UCUAU UUCUAAGAAU UAGAC UUCU UAUU 4444 CUAGA 240 UACUC 354 GUCUC UCAAG UUGAG AGACUAGUA CUAG 4653 AAGCA 241 UGAUA 355 UCCCC CAUUG AAUGU GGGAU AUCA GCUU4665 UGUAU 242 UCAUC 356 CAGUU UCACA GUGAG ACUGA AUGA UACA 4787 ACUAG243 UUACU 357 CACUU GCCCA GGGCA AGUGC GUAA UAGU 5162 AACUA 244 AUGAU 358AACUC GAUAG UAUCA AGUUU UCAU AGUU 5261 GGCCU 245 ACUAA 359 AAAAU UAGGACCUAU UUUUA UAGU GGCC 5270 UCCUA 246 UGUUU 360 UUAGU AAGCA GCUUA CUAAUAACA AGGA 5272 CUAUU 247 UCUGU 361 AGUGC UUAAG UUAAA CACUA CAGA AUAG5338 UGAUA 248 AAGGA 362 UAUCU CCCAA UGGGU GAUAU CCUU AUCA 5737 GCUGU249 AAAUG 363 UAACG GAAAC UUUCC GUUAA AUUU CAGC 5739 UGUUA 250 UGAAA 364ACGUU UGGAA UCCAU ACGUU UUCA AACA 6019 CAGAG 251 UGGAU 365 GUACA UAAAUUUUAA GUACC UCCA UCUG 6059 GGACC 252 UGAAU 366 AGCUA CUCAU UGAGA AGCUGUUCA GUCC 6140 GGGAU 253 UGCCU 367 UAUUC CAUGG CAUGA AAUAA GGCA UCCC6431 CUCCA 254 UAUAG 368 AGCUG AAUAC UAUUC AGCUU UAUA GGAG 6720 UGUAC255 UAUGC 369 CAGAC CACCG GGUGG UCUGG CAUA UACA

Evaluation of Selected Atrogin-1 siRNAs in Transfected Mouse C2C12Myoblasts, Mouse C2C12 Myotubes, Pre-Differentiated Myotubes of PrimaryHuman Skeletal Muscle Cells, and Human SJCRH30 RhabdomyosarcomaMyoblasts.

From the 62 identified siRNAs targeting mouse atrogin-1 and 52 targetinghuman atrogin-1, 30 and 20 siRNAs were selected for synthesis andfunctional analysis, respectively. The activity of these siRNAs wasanalyzed in transfected mouse C2C12 myoblasts, mouse C2C12 myotubes,pre-differentiated myotubes of primary human skeletal muscle cells, andhuman SJCRH30 rhabdomyosarcoma myoblasts.

None of the tested siRNAs targeting mouse atrogin-1 showed significantactivity in mouse C2C12 myotubes (at 10 nM), however 3 siRNAsdownregulated mouse atrogin-1 mRNA by >75% in C2C12 myoblasts (Table 3).In contrast, siRNAs targeting Murf1, which is exclusively expressed inC2C12 myotubes (FIG. 8), were active in C2C12 myotubes, demonstratingthat siRNAs can be transfected into C2C12 myotubes. To determine whetheratrogin-1 might be alternatively spliced in C2C12 myoblasts andmyotubes, various positions in the atrogin-1 mRNA were probed byRT-qPCR, but yielded similar results. Among the 20 tested siRNAstargeting human atrogin-1 only four yielded >75% KD. For both, mouse andhuman atrogin-1, active siRNAs localized either within or close to thecoding region. One of the siRNAs targeting mouse atrogin-1 (1179) wasstrongly cross-reactive with human atrogin-1. While this siRNA failed toshow significant activity in mouse C2C12 myotubes, it effectivelydownregulated human atrogin-1 in myotubes of primary human skeletalmuscle cells. All efficacious siRNAs downregulated their respectivetargets with subnanomolar potency.

Table 3 illustrates activity of selected atrogin-1 siRNAs in transfectedmouse C2C12 myoblasts, mouse C2C12 myotubes, pre-differentiated myotubesof primary human skeletal muscle cells, and human SJCRH30rhabdomyosarcoma myoblasts. For experimental procedures see Example 2.

mu atrogin-1 muC2C12 muC2C12 muC2C12 huSkMC huSkMC huSJCRH30 huSJCRH30NM_026346.3 myotubes myoblasts myoblasts myotubes myotubes myoblastsmyoblasts ID# % KD (10 nM) % KD IC50(nM) % KD IC50(nM) % KD IC50 (nM) 8no KD no KD 499 8.5 34.4 553 9.7 10.6 590 10.1 30.5 0.628 62.8 631 14.483.7 0.159 81.5 0.129 54.5 0.614 694 10.2 50.5 1.228 72.4 0.011 64.60.004 772 8.8 64.7 0.872 0.084 1179 7.2 76.6 0.160 88.5 0.010 86.0 0.0151256 2.7 32.5 1260 no KD 13.6 1459 16.1 60.8 0.258 24.4 1.714 1504 12.876.8 0.092 29.1 0.433 17.3 0.423 1880 14.3 58.6 0.192 66.5 1884 7.7 54.60.135 54.8 0.002 2572 13.7 61.5 0.928 16.0 2.664 18.4 0.027 3007 14.432.7 3668 1.4 6.9 3715 0.7 17.2 3856 no KD 5.9 4139 2.7 10.5 4567 12.856.1 1.589 0.0 37.2 1.028 4673 11.6 34.9 4970 15.6 35.7 5292 20.3 49.60.106 19.3 0.441 11.2 0.02 5640 13.6 40.4 6000 19.7 21.2 6530 3.5 no KD6608 7.4 no KD 6720 17.5 no KD 6799 15.4 no KD 586 66.1 0.326 89.9 0.00890.6 0.011 1071 0.0 no KD 55.9 1077 14.4 1.774 93.9 0.009 93.9 0.0161083 no KD no KD 92.8 0.047 92.2 0.056 1361 no KD 80.1 0.003 81.4 0.1181566 49.9 1679 15.1 1.471 55.8 2582 46.2 2663 no KD no KD 64.2 2999 noKD no KD 55.0 4036 no KD 0.200 64.7 4059 3.2 4117 24.2 1.541 68.0 516215.7 5261 44.4 5272 47.4 5737 no KD no KD 60.8 6019 44.4 6059 no KD noKD 57.6 6431 no KD no KD 65.0

Example 5. In Vitro Screen: MuRF-1

Identification of siRNAs Targeting Mouse Murf1 (Trim63) and/or Human/NHPMuRF1 (TRIM63)

A bioinformatics screen was conducted and identified 51 siRNAs (19mers)that bind specifically to mouse Murf1 sequences that show >3 sequencederivations from mouse Murf2 (Trim55; NM_001039048.2) or Murf3 (Trim54).In addition, 9 siRNAs were identified that target mouse Murf1 and humanMuRF1 (TRIM63; NM_032588.3). A screen for siRNAs (19mers) targetingspecifically human and NHP MuRF1 (NM_032588.3) yielded 52 candidates(Table 4A-Table 41B). All selected siRNA target sites do not harbor SNPs(pos. 2-18).

Tables 4A and 4B illustrate identified siRNA candidates for theregulation of mouse and human/NHP MuRF1.

TABLE 4A sequence of total 23mer SEQ 19mer pos. in target site in IDNM_001039048.2 exon # Mouse mouse × human NM_001039048.2 NO: 33 1 xGAGGAUCCGAGUGGGUUUGGAGA 370 82 1 x CGAGACAGUCGCAUUUCAAAGCA 371 109 1 xGGAUUAUAAAUCUAGCCUGAUUC 372 130 1 x UCCUGAUGGAAACGCUAUGGAGA 373 264 2 xAGGCUGCGAAUCCCUACUGGACC 374 318 2 x GUCGUUUCCGUUGCCCCUCGUGC 375 328 2 xUUGCCCCUCGUGCCGCCAUGAAG 376 329 2 x UGCCCCUCGUGCCGCCAUGAAGU 377 330 2 xGCCCCUCGUGCCGCCAUGAAGUG 378 337 2 x GUGCCGCCAUGAAGUGAUCAUGG 379 346 2 xUGAAGUGAUCAUGGACCGGCACG 380 423 3-Feb x X AGCAGGAGUGCUCCAGUCGGCCC 381457 3 x CAGCCACCCGAUGUGCAAGGAAC 382 460 3 x CCACCCGAUGUGCAAGGAACACG 383495 3 x X UCAACAUCUACUGUCUCACGUGU 384 497 3 x AACAUCUACUGUCUCACGUGUGA385 499 3 x X CAUCUACUGUCUCACGUGUGAGG 386 500 3 x XAUCUACUGUCUCACGUGUGAGGU 387 502 3 x X CUACUGUCUCACGUGUGAGGUGC 388 505 3x X CUGUCUCACGUGUGAGGUGCCUA 389 507 3 x GUCUCACGUGUGAGGUGCCUACU 390 5113 x CACGUGUGAGGUGCCUACUUGCU 391 538 3 x GUGCAAGGUGUUUGGGGCUCACC 392 6094 x CUGAGCUGAGUAACUGCAUCUCC 393 616 4 x GAGUAACUGCAUCUCCAUGCUGG 394 6464 x CAACGACCGAGUGCAGACGAUCA 395 651 4 x ACCGAGUGCAGACGAUCAUCUCU 396 7875 x X GCUGCAGCGGAUCACGCAGGAGC 397 790 5 x GCAGCGGAUCACGCAGGAGCAGG 398911 5 x GAGCCCGGAGGGGCUACCUUCCU 399 1012 7 x UGAGAACAUGGACUACUUUACUC 4001016 7 x AACAUGGACUACUUUACUCUGGA 401 1018 7 x CAUGGACUACUUUACUCUGGACU402 1022 7 x GACUACUUUACUCUGGACUUAGA 403 1130 8 xGAGGAAGAGGGCGUGACCACAGA 404 1266 9 x UACAAUAGGGAAGUGUGUCUUCU 405 1351 9x ACACAAUUGGAAAUGUAUCCAAA 406 1364 9 x UGUAUCCAAAACGUCACAGGACA 407 13669 x UAUCCAAAACGUCACAGGACACU 408 1369 9 x CCAAAACGUCACAGGACACUUUU 4091380 9 x CAGGACACUUUUCUACGUUGGUG 410 1386 9 x ACUUUUCUACGUUGGUGCGAAAU411 1387 9 x CUUUUCUACGUUGGUGCGAAAUG 412 1390 9 xUUCUACGUUGGUGCGAAAUGAAA 413 1391 9 x UCUACGUUGGUGCGAAAUGAAAU 414 1393 9x UACGUUGGUGCGAAAUGAAAUAU 415 1397 9 x UUGGUGCGAAAUGAAAUAUUUUG 416 14549 x X UAUAUGUAUGCCAAUUUGGUGCU 417 1458 9 x X UGUAUGCCAAUUUGGUGCUUUUU 4181460 9 x UAUGCCAAUUUGGUGCUUUUUGU 419 1462 9 x UGCCAAUUUGGUGCUUUUUGUAC420 1466 9 x AAUUUGGUGCUUUUUGUACGAGA 421 1478 9 xUUUGUACGAGAACUUUUGUAUGA 422 1480 9 x UGUACGAGAACUUUUGUAUGAUC 423 1481 9x GUACGAGAACUUUUGUAUGAUCA 424 1483 9 x ACGAGAACUUUUGUAUGAUCACG 425 15209 x GACUGGCGAUUGUCACAAAGUGG 426 1658 9 x GGAUAGGACUGAAUUUGUGUUAU 4271660 9 x AUAGGACUGAAUUUGUGUUAUAU 428 sequence of total 23mer19mer pos. in human + target site in NM_032588.3 NHP NM_032588.3 28 XGGAAGCCAACAGGAUCCGACCCG 429 75 X CCCAGGUCUACUUAGAGCAAAGU 430 77 XCAGGUCUACUUAGAGCAAAGUUA 431 153 X AGUCGAGCCUGAUCCAGGAUGGG 432 239 XCCAGUGGUCAUCUUGCCGUGCCA 433 245 X GUCAUCUUGCCGUGCCAGCACAA 434 248 XAUCUUGCCGUGCCAGCACAACCU 435 249 X UCUUGCCGUGCCAGCACAACCUG 436 259 XCCAGCACAACCUGUGCCGGAAGU 437 339 X UGUCCAUGUCUGGAGGCCGUUUC 438 367 XCCCCACCUGCCGCCACGAGGUGA 439 368 X CCCACCUGCCGCCACGAGGUGAU 440 370 XCACCUGCCGCCACGAGGUGAUCA 441 371 X ACCUGCCGCCACGAGGUGAUCAU 442 372 XCCUGCCGCCACGAGGUGAUCAUG 443 373 X CUGCCGCCACGAGGUGAUCAUGG 444 374 XUGCCGCCACGAGGUGAUCAUGGA 445 375 X GCCGCCACGAGGUGAUCAUGGAU 446 379 XCCACGAGGUGAUCAUGGAUCGUC 447 380 X CACGAGGUGAUCAUGGAUCGUCA 448 381 XACGAGGUGAUCAUGGAUCGUCAC 449 384 X AGGUGAUCAUGGAUCGUCACGGA 450 385 XGGUGAUCAUGGAUCGUCACGGAG 451 386 X GUGAUCAUGGAUCGUCACGGAGU 452 387 XUGAUCAUGGAUCGUCACGGAGUG 453 451 X CAUCUACAAACAGGAGUGCUCCA 454 458 XAAACAGGAGUGCUCCAGUCGGCC 455 459 X AACAGGAGUGCUCCAGUCGGCCG 456 461 XCAGGAGUGCUCCAGUCGGCCGCU 457 491 X GGCAGUCACCCCAUGUGCAAGGA 458 499 XCCCCAUGUGCAAGGAGCACGAAG 459 503 X AUGUGCAAGGAGCACGAAGAUGA 460 531 XUCAACAUCUACUGUCUCACGUGU 461 535 X CAUCUACUGUCUCACGUGUGAGG 462 539 XUACUGUCUCACGUGUGAGGUGCC 463 564 X CCUGCUCCAUGUGCAAGGUGUUU 464 568 XCUCCAUGUGCAAGGUGUUUGGGA 465 610 X GGCCCCAUUGCAGAGUGUCUUCC 466 612 XCCCCAUUGCAGAGUGUCUUCCAG 467 645 X CUGAACUGAAUAACUGUAUCUCC 468 647 XGAACUGAAUAACUGUAUCUCCAU 469 670 X GCUGGUGGCGGGGAAUGACCGUG 470 671 XCUGGUGGCGGGGAAUGACCGUGU 471 672 X UGGUGGCGGGGAAUGACCGUGUG 472 673 XGGUGGCGGGGAAUGACCGUGUGC 473 812 X AAAAGUGAGUUGCUGCAGCGGAU 474 860 XAGCUUCAUCGAGGCCCUCAUCCA 475 968 X CUCUUGACUGCCAAGCAACUCAU 476 970 XCUUGACUGCCAAGCAACUCAUCA 477 977 X GCCAAGCAACUCAUCAAAAGCAU 478 979 XCAAGCAACUCAUCAAAAGCAUUG 479 980 X AAGCAACUCAUCAAAAGCAUUGU 480

TABLE 4B SEQ SEQ exon ID ID # NO: NO: 19mer pos. in sense antisenseNM_001 strand strand 103 sequence sequence 9048.2 (5′-3′) (5′-3′) 33 1GGAUC 481 UCCAA 592 CGAGU ACCCA GGGUU CUCGG UGGA AUCC 82 1 AGACA 482CUUUG 593 GUCGC AAAUG AUUUC CGACU AAAG GUCU 109 1 AUUAU 483 AUCAG 594AAAUC GCUAG UAGCC AUUUA UGAU UAAU 130 1 CUGAU 484 UCCAU 595 GGAAA AGCGUCGCUA UUCCA UGGA UCAG 264 2 GCUGC 485 UCCAG 596 GAAUC UAGGG CCUAC AUUCGUGGA CAGC 318 2 CGUUU 486 ACGAG 597 CCGUU GGGCA GCCCC ACGGA UCGU AACG328 2 GCCCC 487 UCAUG 598 UCGUG GCGGC CCGCC ACGAG AUGA GGGC 329 2 CCCCU488 UUCAU 599 CGUGC GGCGG CGCCA CACGA UGAA GGGG 330 2 CCCUC 489 CUUCA600 GUGCC UGGCG GCCAU GCACG GAAG AGGG 337 2 GCCGC 490 AUGAU 601 CAUGACACUU AGUGA CAUGG UCAU CGGC 346 2 AAGUG 491 UGCCG 602 AUCAU GUCCA GGACCUGAUC GGCA ACUU 423 3- CAGGA 492 GCCGA 603 Feb GUGCU CUGGA CCAGU GCACUCGGC CCUG 457 3 GCCAC 493 UCCUU 604 CCGAU GCACA GUGCA UCGGG AGGA UGGC460 3 ACCCG 494 UGUUC 605 AUGUG CUUGC CAAGG ACAUC AACA GGGU 495 3 AACAU495 ACGUG 606 CUACU AGACA GUCUC GUAGA ACGU UGUU 497 3 CAUCU 496 ACACG607 ACUGU UGAGA CUCAC CAGUA GUGU GAUG 499 3 UCUAC 497 UCACA 608 UGUCUCGUGA CACGU GACAG GUGA UAGA 500 3 CUACU 498 CUCAC 609 GUCUC ACGUG ACGUGAGACA UGAG GUAG 502 3 ACUGU 499 ACCUC 610 CUCAC ACACG GUGUG UGAGA AGGUCAGU 505 3 GUCUC 500 GGCAC 611 ACGUG CUCAC UGAGG ACGUG UGCC AGAC 507 3CUCAC 501 UAGGC 612 GUGUG ACCUC AGGUG ACACG CCUA UGAG 511 3 CGUGU 502CAAGU 613 GAGGU AGGCA GCCUA CCUCA CUUG CACG 538 3 GCAAG 503 UGAGC 614GUGUU CCCAA UGGGG ACACC CUCA UUGC 609 4 GAGCU 504 AGAUG 615 GAGUA CAGUUACUGC ACUCA AUCU GCUC 616 4 GUAAC 505 AGCAU 616 UGCAU GGAGA CUCCA UGCAGUGCU UUAC 646 4 ACGAC 506 AUCGU 617 CGAGU CUGCA GCAGA CUCGG CGAU UCGU651 4 CGAGU 507 AGAUG 618 GCAGA AUCGU CGAUC CUGCA AUCU CUCG 787 5 UGCAG508 UCCUG 619 CGGAU CGUGA CACGC UCCGC AGGA UGCA 790 5 AGCGG 509 UGCUC620 AUCAC CUGCG GCAGG UGAUC AGCA CGCU 911 5 GCCCG 510 GAAGG 621 GAGGGUAGCC GCUAC CCUCC CUUC GGGC 1012 7 AGAAC 511 GUAAA 622 AUGGA GUAGU CUACUCCAUG UUAC UUCU 1016 7 CAUGG 512 CAGAG 623 ACUAC UAAAG UUUAC UAGUC UCUGCAUG 1018 7 UGGAC 513 UCCAG 624 UACUU AGUAA UACUC AGUAG UGGA UCCA 1022 7CUACU 514 UAAGU 625 UUACU CCAGA CUGGA GUAAA CUUA GUAG 1130 8 GGAAG 515UGUGG 626 AGGGC UCACG GUGAC CCCUC CACA UUCC 1266 9 CAAUA 516 AAGAC 627GGGAA ACACU GUGUG UCCCU UCUU AUUG 1351 9 ACAAU 517 UGGAU 628 UGGAA ACAUUAUGUA UCCAA UCCA UUGU 1364 9 UAUCC 518 UCCUG 629 AAAAC UGACG GUCAC UUUUGAGGA GAUA 1366 9 UCCAA 519 UGUCC 630 AACGU UGUGA CACAG CGUUU GACA UGGA1369 9 AAAAC 520 AAGUG 631 GUCAC UCCUG AGGAC UGACG ACUU UUUU 1380 9GGACA 521 CCAAC 632 CUUUU GUAGA CUACG AAAGU UUGG GUCC 1386 9 UUUUC 522UUCGC 633 UACGU ACCAA UGGUG CGUAG CGAA AAAA 1387 9 UUUCU 523 UUUCG 634ACGUU CACCA GGUGC ACGUA GAAA GAAA 1390 9 CUACG 524 UCAUU 635 UUGGU UCGCAGCGAA CCAAC AUGA GUAG 1391 9 UACGU 525 UUCAU 636 UGGUG UUCGC CGAAA ACCAAUGAA CGUA 1393 9 CGUUG 526 AUUUC 637 GUGCG AUUUC AAAUG GCACC AAAU AACG1397 9 GGUGC 527 AAAUA 638 GAAAU UUUCA GAAAU UUUCG AUUU CACC 1454 9UAUGU 528 CACCA 639 AUGCC AAUUG AAUUU GCAUA GGUG CAUA 1458 9 UAUGC 529AAAGC 640 CAAUU ACCAA UGGUG AUUGG CUUU CAUA 1460 9 UGCCA 530 AAAAA 641AUUUG GCACC GUGCU AAAUU UUUU GGCA 1462 9 CCAAU 531 ACAAA 642 UUGGU AAGCAGCUUU CCAAA UUGU UUGG 1466 9 UUUGG 532 UCGUA 643 UGCUU CAAAA UUUGU AGCACACGA CAAA 1478 9 UGUAC 533 AUACA 644 GAGAA AAAGU CUUUU UCUCG GUAU UACA1480 9 UACGA 534 UCAUA 645 GAACU CAAAA UUUGU GUUCU AUGA CGUA 1481 9ACGAG 535 AUCAU 646 AACUU ACAAA UUGUA AGUUC UGAU UCGU 1483 9 GAGAA 536UGAUC 647 CUUUU AUACA GUAUG AAAGU AUCA UCUC 1520 9 CUGGC 537 ACUUU 648GAUUG GUGAC UCACA AAUCG AAGU CCAG 1658 9 AUAGG 538 AACAC 649 ACUGA AAAUUAUUUG CAGUC UGUU CUAU 1660 9 AGGAC 539 AUAAC 650 UGAAU ACAAA UUGUG UUCAGUUAU UCCU 19mer pos. sense antisense in strand strand NM_032 sequencesequence 588.3 (5′-3′) (5′-3′) 28 AAGCC 540 GGUCG 651 AACAG GAUCC GAUCCUGUUG GACC GCUU 75 CAGGU 541 UUUGC 652 CUACU UCUAA UAGAG GUAGA CAAA CCUG77 GGUCU 542 ACUUU 653 ACUUA GCUCU GAGCA AAGUA AAGU GACC 153 UCGAG 543CAUCC 654 CCUGA UGGAU UCCAG CAGGC GAUG UCGA 239 AGUGG 544 GCACG 655UCAUC GCAAG UUGCC AUGAC GUGC CACU 245 CAUCU 545 GUGCU 656 UGCCG GGCACUGCCA GGCAA GCAC GAUG 248 CUUGC 546 GUUGU 657 CGUGC GCUGG CAGCA CACGGCAAC CAAG 249 UUGCC 547 GGUUG 658 GUGCC UGCUG AGCAC GCACG AACC GCAA 259AGCAC 548 UUCCG 659 AACCU GCACA GUGCC GGUUG GGAA UGCU 339 UCCAU 549AACGG 660 GUCUG CCUCC GAGGC AGACA CGUU UGGA 367 CCACC 550 ACCUC 661UGCCG GUGGC CCACG GGCAG AGGU GUGG 368 CACCU 551 CACCU 662 GCCGC CGUGGCACGA CGGCA GGUG GGUG 370 CCUGC 552 AUCAC 663 CGCCA CUCGU CGAGG GGCGGUGAU CAGG 371 CUGCC 553 GAUCA 664 GCCAC CCUCG GAGGU UGGCG GAUC GCAG 372UGCCG 554 UGAUC 665 CCACG ACCUC AGGUG GUGGC AUCA GGCA 373 GCCGC 555AUGAU 666 CACGA CACCU GGUGA CGUGG UCAU CGGC 374 CCGCC 556 CAUGA 667ACGAG UCACC GUGAU UCGUG CAUG GCGG 375 CGCCA 557 CCAUG 668 CGAGG AUCACUGAUC CUCGU AUGG GGCG 379 ACGAG 558 CGAUC 669 GUGAU CAUGA CAUGG UCACCAUCG UCGU 380 CGAGG 559 ACGAU 670 UGAUC CCAUG AUGGA AUCAC UCGU CUCG 381GAGGU 560 GACGA 671 GAUCA UCCAU UGGAU GAUCA CGUC CCUC 384 GUGAU 561CGUGA 672 CAUGG CGAUC AUCGU CAUGA CACG UCAC 385 UGAUC 562 CCGUG 673AUGGA ACGAU UCGUC CCAUG ACGG AUCA 386 GAUCA 563 UCCGU 674 UGGAU GACGACGUCA UCCAU CGGA GAUC 387 AUCAU 564 CUCCG 675 GGAUC UGACG GUCAC AUCCAGGAG UGAU 451 UCUAC 565 GAGCA 676 AAACA CUCCU GGAGU GUUUG GCUC UAGA 458ACAGG 566 CCGAC 677 AGUGC UGGAG UCCAG CACUC UCGG CUGU 459 CAGGA 567GCCGA 678 GUGCU CUGGA CCAGU GCACU CGGC CCUG 461 GGAGU 568 CGGCC 679GCUCC GACUG AGUCG GAGCA GCCG CUCC 491 CAGUC 569 CUUGC 680 ACCCC ACAUGAUGUG GGGUG CAAG ACUG 499 CCAUG 570 UCGUG 681 UGCAA CUCCU GGAGC UGCACACGA AUGG 503 GUGCA 571 AUCUU 682 AGGAG CGUGC CACGA UCCUU AGAU GCAC 531AACAU 572 ACGUG 683 CUACU AGACA GUCUC GUAGA ACGU UGUU 535 UCUAC 573UCACA 684 UGUCU CGUGA CACGU GACAG GUGA UAGA 539 CUGUC 574 CACCU 685UCACG CACAC UGUGA GUGAG GGUG ACAG 564 UGCUC 575 ACACC 686 CAUGU UUGCAGCAAG CAUGG GUGU AGCA 568 CCAUG 576 CCAAA 687 UGCAA CACCU GGUGU UGCACUUGG AUGG 610 CCCCA 577 AAGAC 688 UUGCA ACUCU GAGUG GCAAU UCUU GGGG 612CCAUU 578 GGAAG 689 GCAGA ACACU GUGUC CUGCA UUCC AUGG 645 GAACU 579AGAUA 690 GAAUA CAGUU ACUGU AUUCA AUCU GUUC 647 ACUGA 580 GGAGA 691AUAAC UACAG UGUAU UUAUU CUCC CAGU 670 UGGUG 581 CGGUC 692 GCGGG AUUCCGAAUG CCGCC ACCG ACCA 671 GGUGG 582 ACGGU 693 CGGGG CAUUC AAUGA CCCGCCCGU CACC 672 GUGGC 583 CACGG 694 GGGGA UCAUU AUGAC CCCCG CGUG CCAC 673UGGCG 584 ACACG 695 GGGAA GUCAU UGACC UCCCC GUGU GCCA 812 AAGUG 585CCGCU 696 AGUUG GCAGC CUGCA AACUC GCGG ACUU 860 CUUCA 586 GAUGA 697UCGAG GGGCC GCCCU UCGAU CAUC GAAG 968 CUUGA 587 GAGUU 698 CUGCC GCUUGAAGCA GCAGU ACUC CAAG 970 UGACU 588 AUGAG 699 GCCAA UUGCU GCAAC UGGCAUCAU GUCA 977 CAAGC 589 GCUUU 700 AACUC UGAUG AUCAA AGUUG AAGC CUUG 979AGCAA 590 AUGCU 701 CUCAU UUUGA CAAAA UGAGU GCAU UGCU 980 GCAAC 591AAUGC 702 UCAUC UUUUG AAAAG AUGAG CAUU UUGC

Activity of Selected MuRF1 SIRNAS in Transfected Mouse C2C12 Myotubesand Pre-Differentiated Myotubes of Primary Human Skeletal Muscle Cells

From the 60 identified siRNAs targeting mouse Murf1 and 25 siRNAstargeting human MuRF1, 35 and 25 siRNAs were selected for synthesis,respectively. The activity of these siRNAs was analyzed in transfectedmouse C2C12 myotubes and pre-differentiated primary human skeletalmuscle cells (Table 5). Among the siRNAs targeting mouse MurF1, 14displayed >70% knock down of Murf1, but <20% knock down of Murf2 andMurf3 in C2C12 myotubes. At least 6 of these 14 siRNAs were crossreactive with human MuRF1. Among the tested siRNAs targeting humanMuRF1, 8 displayed >70% knock down of MuRF1, but <20% knock down ofMuRF2 and MuRF3 in pre-differentiated myotubes of primary human skeletalmuscle cells. Only 1 of these 8 siRNAs showed significantcross-reactivity with mouse Muf1. All efficacious siRNAs downregulatedtheir respective targets with subnanomolar potency.

Table 5 illustrates activity of selected MuRF1 siRNAs in transfectedmouse C2C12 myotubes and pre-differentiated myotubes of primary humanskeletal muscle cells. Cells were grown and transfected and RNAsisolated and analyzed as described in Example 5.

muC2C12 muC2C12 muC2C12 muC2C12 huSkMC huSkMC huSkMC huSkMC 19mermyotubes myotubes myotubes myotubes myotubes myotubes myotubes myotubesposition in mMuRF1 mMuRF2 mMuRF3 mMuRF1 hMuRF1 hMuRF2 hMuRF3 hMuRf1NM_026346.3 KD(%) KD(%) KD(%) IC50(nM) KD(%) KD(%) KD(%) IC50(nM) 3324.3 0.0 5.5 109 73.4 0.0 6.0 0.073 130 45.8 10.4 16.0 264 79.6 3.7 22.00.172 80.9 5.6 0.0 0.018 337 42.9 10.4 7.9 423 65.9 15.5 8.9 0.288 64.5460 56.4 16.6 17.8 495 70.7 9.6 31.9 0.495 76.1 49.0 4.4 0.105 499 73.812.7 7.0 0.116 76.0 11.3 24.0 0.150 500 69.0 20.7 11.5 0.167 92.5 23.350.0 538 48.6 10.7 16.5 651 78.3 5.8 0.0 0.082 53.9 0.0 4.2 0.150 78726.0 13.5 7.9 911 43.5 0.0 10.0 1012 74.1 0.0 27.5 0.014 83.1 6.6 0.00.019 1018 72.6 12.3 6.3 0.121 1022 70.9 26.5 22.9 0.042 1130 60.6 22.016.3 0.206 1266 73.5 27.1 19.6 0.007 83.8 7.5 42.3 0.184 1351 77.5 44.20.0 0.008 79.0 6.6 52.4 0.509 1364 71.6 0.8 2.8 0.012 27.9 1387 79.1 9.514.9 0.007 66.7 15.0 13.3 0.059 1390 73.6 33.1 10.0 0.012 1393 75.2 28.44.5 0.044 73.3 340 12.1 0 141 1397 78.8 5.2 16.4 0.008 74.9 23.0 12.30.009 1454 73.8 6.5 8.7 0.019 68.2 7.0 1.7 0.004 1458 73.1 0.0 6.4 0.01685.8 13.7 42.7 0.008 1462 69.9 17.2 19.8 0.017 63.8 3.8 0.0 0.005 146675.0 17.8 19.1 0.012 68.4 6.2 0.0 0.045 1480 71.1 11.9 6.9 0.022 148165.8 12.2 0.4 0.266 1483 74.6 1.1 4.0 0.030 1520 72.8 1.6 8.8 0.012 30.61658 73.7 21.6 9.8 0.028 26.0 15.6 6.6 0.005 1660 76.2 0.0 0.0 0.017 7525.8 69.6 0.0 0.0 77 14.5 17.4 13.8 83.7 7.1 13.9 0.152 245 60.3 0.90570.9 33.1 14.3 259 49.7 2.759 75.1 84.5 71.1 0.053 339 52.7 1.2 14.3 3670.0 0.0 0.0 370 8.8 0.033 21.2 25.0 4.1 373 64.6 21.8 14.1 374 33.7 7.014.8 0.659 89.7 10.0 19.7 0.110 380 19.5 0.013 70.6 8.5 0.4 386 69.919.9 16.4 0.002 459 66.7 0.148 78.1 25.2 7.1 491 57.1 16.3 1.3 503 56.40.101 52.0 89.9 86.1 535 70.8 32.4 13.4 0.074 82.1 24.0 9.5 0.008 5648.2 0.002 72.4 26.1 0.6 610 6.5 18.7 17.0 89.6 9.4 15.2 0.107 645 46.213.9 2.3 3.475 85.2 0.0 0.0 0.007 647 77.5 20.1 0.0 0.211 94.6 4.4 19.40.006 673 35.5 13.4 4.5 860 77.1 22.5 0.0 970 8.8 29.3 10.6 84.9 12.97.3 0.056 977 19.9 5.9 0.0 2.838 93.6 51.0 12.6 0.117 979 87.4 29.6 17.40.058 980 0.0 36.0 2.1 93.6 4.7 20.5 0.118

Example 6. 2017-PK-279-WT—CD71 vs IgG2A Isotype, HPRT Vs MSTN siRNADesign and Synthesis

MSTN: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against mouse MSTN. The sequence (5′to 3′) of the guide/antisense strand was UUAUUAUUUGUUCUUUGCCUU (SEQ IDNO: 14226). Base, sugar and phosphate modifications were used tooptimize the potency of the duplex and reduce immunogenicity. All siRNAsingle strands were fully assembled on solid phase using standardphospharamidite chemistry and purified over HPLC. Purified singlestrands were duplexed to get the double stranded siRNA. The passengerstrand contained two conjugation handles, a C6-NH₂ at the 5′ end and aC6-SH at the 3′ end. Both conjugation handles were connected to siRNApassenger strand via phosphorothioate-inverted abasic-phosphorothioatelinker.

HPRT: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against mouse MSTN. The sequence (5′to 3′) of the guide/antisense strand was UUAAAAUCUACAGUCAUAGUU (SEQ IDNO: 14227). Base, sugar and phosphate modifications were used tooptimize the potency of the duplex and reduce immunogenicity. All siRNAsingle strands were fully assembled on solid phase using standardphospharamidite chemistry and purified over HPLC. Purified singlestrands were duplexed to get the double stranded siRNA. The passengerstrand contained two conjugation handles, a C6-NH₂ at the 3′ end and aC6-SH at the 5′ end. Both conjugation handles were connected to siRNApassenger strand via phosphorothioate-inverted abasic-phosphorothioatelinker.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 14228). The same base, sugar and phosphate modifications that wereused for the active MSTN siRNA duplex were used in the negative controlsiRNA. All siRNA single strands were fully assembled on solid phaseusing standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker.

ASC Synthesis and Characterization

The CD71 mAb-siRNA DAR1 conjugates were made, purified and characterizedas described in Example 3. All conjugates were made through cysteineconjugation, a SMCC linker and the PEG was attached at the thiol usingarchitecture 1 for MSTN and the scrambled siRNA and architecture 2 forthe HPRT siRNA, see Example 3. Conjugates were characterizedchromatographically as described in Table 6.

TABLE 6 HPLC retention time (RT) in minutes RT, SAX RT, SEC GroupsConjugate Method-2 Method-1  1-4 TfR-mAb-HPRT-PEG5k; DAR1 8.8 7.1  5-8IgG2a-mAb-HPRT-PEG5k; DAR1 8.9 7.7  9-12 TfR-mAb-MSTN-PEG5k; DAR1 8.77.2 13-16 IgG2a-mAb-MSTN-PEG5k; DAR1 8.9 7.7 17-20TfR-mAb-scrambled-PEG5k; DAR1 8.4 7.2

In Vivo Study Design

The conjugates were assessed for their ability to mediate mRNAdownregulation of myostatin (MSTN) in skeletal muscle in vivo in wildtype CD-1 mice. Mice were dosed via intravenous (iv) injection with PBSvehicle control and the indicated ASCs and doses, see FIG. 9A. After 96hours, gastrocnemius (gastroc) muscle tissues were harvested andsnap-frozen in liquid nitrogen. mRNA knockdown in target tissue wasdetermined using a comparative qPCR assay. Total RNA was extracted fromthe tissue, reverse transcribed and mRNA levels were quantified usingTaqMan qPCR, using the appropriately designed primers and probes. PPIB(housekeeping gene) was used as an internal RNA loading control, resultswere calculated by the comparative Ct method, where the differencebetween the target gene Ct value and the PPIB Ct value (ΔCt) iscalculated and then further normalized relative to the PBS control groupby taking a second difference (ΔΔCt).

Results

For gastrocnemius muscle harvested 96 hours post-dose, maximum MSTN mRNAdownregulation of greater than 90% was observed after a singleintravenous dose of 3 mg/kg of siRNA, see FIG. 9B. In addition, a doseresponse was also observed (dose range: 0.3 to 3.0 mg/kg siRNA) and nosignificant mRNA downregulation was observed for the control groups.

Conclusions

In gastrocnemius muscle, it was demonstrated that an ASC is able todownregulate a muscle specific gene. The ASC was made with ananti-transferrin antibody conjugated to an siRNA designed to downregulate MSTN mRNA. Mouse gastroc muscle expresses the transferrinreceptor and the conjugate has a mouse specific anti-transferrinantibody to target the siRNA, resulting in accumulation of theconjugates in gastroc muscle. Receptor mediate uptake resulted in siRNAmediated knockdown of the MSTN mRNA.

Example 7. 2017-PK-289-WT—CD71 mAb MSTN Time Course for Phenotype siRNADesign and Synthesis

MSTN: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against mouse MSTN. The sequence (5′to 3″) of the guide/antisense strand was UUAUUAUUUGUUCUUUGCCUU (SEQ IDNO: 14226). Base, sugar and phosphate modifications were used tooptimize the potency of the duplex and reduce immunogenicity. All siRNAsingle strands were fully assembled on solid phase using standardphospharamidite chemistry and purified over HPLC. Purified singlestrands were duplexed to get the double stranded siRNA. The passengerstrand contained two conjugation handles, a C6-NH₂ at the 5′ end and aC6-SH at the 3′ end. Both conjugation handles were connected to siRNApassenger strand via phosphorothioate-inverted abasic-phosphorothioatelinker. Because the free thiol was not being used for conjugation, itwas end capped with N-ethylmaleimide.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 14228). The same base, sugar and phosphate modifications that wereused for the active MSTN siRNA duplex were used in the negative controlsiRNA. All siRNA single strands were fully assembled on solid phaseusing standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker. Because the free thiol was not beingused for conjugation, it was end capped with N-ethylmaleimide.

ASC Synthesis and Characterization

The CD71 mAb-siRNA DAR1 conjugates were made and characterized asdescribed in Example 3. All conjugates were made through cysteineconjugation, a SMCC linker and the thiol was end capped with NEM usingarchitecture 1. Conjugates were characterized chromatographically asdescribed in Table 7.

TABLE 7 HPLC retention time (RT) in minutes RT, SAX RT, SEC GroupsConjugate Method-2 Method-1 1-4, 13 & 14 TfR-mAb-MSTN-NEM; DAR1 8.7 10.05-8 TfR-mAb-scrambled-NEM; DAR1 8.9 10.0

In Vivo Study Design

The conjugates were assessed for their ability to mediate mRNAdownregulation of myostatin (MSTN) in skeletal muscle in vivo in wildtype CD-1 mice. Mice were dosed via intravenous (iv) injection with PBSvehicle control and the indicated ASCs at the doses indicated in FIG.10A. Plasma and tissue samples were also taken as indicated in FIG. 10A.Muscle tissues were harvested and snap-frozen in liquid nitrogen. mRNAknockdown in target tissue was determined using a comparative qPCR assayas described in the methods section. Total RNA was extracted from thetissue, reverse transcribed and mRNA levels were quantified using TaqManqPCR, using the appropriately designed primers and probes. PPIB(housekeeping gene) was used as an internal RNA loading control, resultswere calculated by the comparative Ct method, where the differencebetween the target gene Ct value and the PPIB Ct value (ΔCt) iscalculated and then further normalized relative to the PBS control groupby taking a second difference (ΔΔCt). Quantitation of tissue siRNAconcentrations was determined using a stem-loop qPCR assay as describedin the methods section. The antisense strand of the siRNA was reversetranscribed using a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves.

Plasma myostatin levels were determined using an ELISA, see Example 2for full experimental details. Changes in leg muscle area weredetermined: The leg-to-be-measured were shaved and a line was drawnusing indelible ink to mark region of measurement. Mice were restrainedin a cone restraint and the right leg was held by hand. Digital caliperswere used to take one measurement on the sagittal plane and another onthe coronal plane. The procedure was repeated twice per week.

Results

Quantifiable levels of siRNA accumulated in muscle tissue after a singleintravenous dose of the antibody siRNA conjugates, see FIG. 10B. RobustMSTN mRNA downregulation was observed in gastrocnemius muscle, whichresulted in a reduction in the levels of MSTN protein in the plasma,after a single intravenous dose of 3 mg/kg of siRNA, see FIG. 10C andFIG. 10D. Maximum mRNA downregulation of ˜90% was observed between 7-14days post-dose. At 6 weeks post-dose gastroc muscle had approximately75% mRNA downregulation, which corresponded to about a 50% reduction inplasma protein levels relative to the PBS or anti-transferrin antibodyconjugated scrambled controls. Downregulation of MSTN resulted instatistically significant increases in muscle size, see FIG. 10E andFIG. 10F.

Conclusions

In this example it was demonstrated that accumulation of siRNA invarious muscle tissues after a single dose of an anti-transferrinantibody targeted siRNA conjugate. In Gastroc muscle, significant andlong-lasting siRNA mediated MSTN mRNA downregulation was observed. Mousegastroc muscle expresses transferrin receptor and the conjugate has amouse specific anti-transferrin antibody to target the siRNA, resultingin accumulation of the conjugates in gastroc muscle. Receptor mediateuptake resulted in siRNA mediated knockdown of the MSTN gene.

Example 8: 2017-PK-299-WT—MSTN Zalu Vs TfR, mAb Vs Fab, DAR1 vs DAR2siRNA Design and Synthesis

MSTN: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against mouse MSTN. The sequence (5′to 3′) of the guide/antisense strand was UUAUUAUUUGUUCUUUGCCUU (SEQ IDNO: 14226). Base, sugar and phosphate modifications were used tooptimize the potency of the duplex and reduce immunogenicity. All siRNAsingle strands were fully assembled on solid phase using standardphospharamidite chemistry and purified over HPLC. Purified singlestrands were duplexed to get the double stranded siRNA. The passengerstrand contained two conjugation handles, a C6-NH₂ at the 5′ end and aC6-SH at the 3′ end. Both conjugation handles were connected to siRNApassenger strand via phosphorothioate-inverted abasic-phosphorothioatelinker. Because the free thiol was not being used for conjugation, itwas end capped with N-ethylmaleimide.

MSTN*: MSTN: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against mouse MSTN. The sequence (5′to 3′) of the guide/antisense strand was UUAUUAUUUGUUCUUUGCCUU (SEQ IDNO: 14226). Base, sugar and phosphate modifications were used tooptimize the potency of the duplex and reduce immunogenicity. All siRNAsingle strands were fully assembled on solid phase using standardphospharamidite chemistry and purified over HPLC. Purified singlestrands were duplexed to get the double stranded siRNA. The passengerstrand contained one conjugation handle, a C6-NH₂ at the 5′ end, whichwas connected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker.

ASC Synthesis and Characterization

The CD71 mAb-siRNA DAR1 and DAR2 conjugates were made and characterizedas described in Example 3. Groups 1-8 and 17-20 were made throughcysteine conjugation and a BisMal linker using architecture 3. Groups9-16 were made through cysteine conjugation, a SMCC linker and the freethiol was end capped with NEM PEG using architecture 1. Conjugates werecharacterized chromatographically as described in Table 8.

TABLE 8 HPLC retention time (RT) in minutes RT, SAX RT, SEC GroupsConjugate Method-2 Method-1  1-4 TfR-Fab-MSTN; DAR1 8.7 10.0  5-8EGFR-Fab-MSTN; DAR1 8.9 10.0  9-12 TfR-mAb-MSTN-NEM; DAR1 9.5 7.9 13-16TfR-mAb-MSTN-nEM; DAR2 10.3 8.1 17-18 EGFR-mAb-MSTN; DAR1 9.3 NT 19-20EGFR-mAb-MSTN; DAR2 10.2 NT

In Vivo Study Design

The conjugates were assessed for their ability to mediate mRNAdownregulation of myostatin (MSTN) in skeletal muscle in vivo in wildtype CD-1 mice. Mice were dosed via intravenous (iv) injection with PBSvehicle control and the indicated ASCs at the doses indicated in FIG.11A. Plasma and tissue samples were also taken as indicated in FIG. 11A.Gastrocnemius (gastroc) muscle tissues were harvested and snap-frozen inliquid nitrogen. mRNA knockdown in target tissue was determined using acomparative qPCR assay as described in the methods section. Total RNAwas extracted from the tissue, reverse transcribed and mRNA levels werequantified using TaqMan qPCR, using the appropriately designed primersand probes. PPIB (housekeeping gene) was used as an internal RNA loadingcontrol, results were calculated by the comparative Ct method, where thedifference between the target gene Ct value and the PPIB Ct value (ΔCt)is calculated and then further normalized relative to the PBS controlgroup by taking a second difference (ΔΔCt). Quantitation of tissue siRNAconcentrations was determined using a stem-loop qPCR assay as describedin the methods section. The antisense strand of the siRNA was reversetranscribed using a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves.

Results

Quantifiable levels of siRNA accumulated in muscle tissue after a singleintravenous dose of the antibody and Fab siRNA conjugates, see FIG. 11B.Robust MSTN mRNA downregulation was observed in gastroc muscle, when theanti-transferrin antibody conjugate was administered as a DAR1 or DAR2,or as a Fab DAR1 conjugate, see FIG. 11C.

Conclusions

In this example it was demonstrated that accumulation of siRNA ingastroc muscle tissue after a single dose of an anti-transferrinantibody and Fab targeted siRNA conjugates. In Gastroc muscle, siRNAmediated MSTN mRNA downregulation with DAR1 and DAR2 antibody conjugateswere observed, in addition to the DAR1 Fab conjugate. Mouse gastrocmuscle expresses transferrin receptor and the conjugate has a mousespecific anti-transferrin antibody or Fab to target the siRNA, resultingin accumulation of the conjugates in gastroc muscle. Receptor mediateuptake resulted in siRNA mediated knockdown of the MSTN gene.

Example 9: 2017-PK-303-WT—Dose Response MSTN mAb Vs Fab Vs Chol siRNADesign and Synthesis

MSTN: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against mouse MSTN. The sequence (5′to 3′) of the guide/antisense strand was UUAUUAUUUGUUCUUUGCCUU (SEQ IDNO: 14226). Base, sugar and phosphate modifications were used tooptimize the potency of the duplex and reduce immunogenicity. All siRNAsingle strands were fully assembled on solid phase using standardphospharamidite chemistry and purified over HPLC. Purified singlestrands were duplexed to get the double stranded siRNA. The passengerstrand contained two conjugation handles, a C6-NH₂ at the 5′ end and aC6-SH at the 3′ end. Both conjugation handles were connected to siRNApassenger strand via phosphorothioate-inverted abasic-phosphorothioatelinker. Because the free thiol was not being used for conjugation, itwas end capped with N-ethylmaleimide.

MSTN*: MSTN: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against mouse MSTN. The sequence (5′to 3′) of the guide/antisense strand was UUAUUAUUUGUUCUUUGCCUU (SEQ IDNO: 14226). Base, sugar and phosphate modifications were used tooptimize the potency of the duplex and reduce immunogenicity. All siRNAsingle strands were fully assembled on solid phase using standardphospharamidite chemistry and purified over HPLC. Purified singlestrands were duplexed to get the double stranded siRNA. The passengerstrand contained one conjugation handle, a C6-NH₂ at the 5′ end, whichwas connected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 14228). The same base, sugar and phosphate modifications that wereused for the active MSTN siRNA duplex were used in the negative controlsiRNA. All siRNA single strands were fully assembled on solid phaseusing standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker. Because the free thiol was not beingused for conjugation, it was end capped with N-ethylmaleimide.

ASC Synthesis and Characterization

The CD71 mAb-siRNA DAR1 and DAR2 conjugates were made and characterizedas described in Example 3. Groups 5-12 made through cysteineconjugation, a BisMal linker using architecture 3. Groups 13-16 weremade through cysteine conjugation, a SMCC linker, the free thiol was endcapped with NEM using architecture 1. Groups 17-20 were made throughcysteine conjugation, a BisMal linker, the free thiol was end cappedwith NEM using architecture 3. Conjugates were characterizedchromatographically as described Table 9.

TABLE 9 HPLC retention time (RT) in minutes RT, SAX RT, SEC GroupsConjugate Method-2 Method-1  5-8 TfR-Fab-MSTN*; DAR1 8.7 10.0  9-12TfR-mAb-MSTN*; DAR1 9.3 7.8 13-16 TfR-mAb-MSTN; DAR1 9.5 7.9 17-20TfR-mAb-scramble; DAR1 9.1 7.3

In Vivo Study Design

The conjugates were assessed for their ability to mediate mRNAdownregulation of myostatin (MSTN) in skeletal muscle in vivo in wildtype CD-1 mice. Mice were dosed via intravenous (iv) injection with PBSvehicle control and the indicated ASCs at the doses indicated in FIG.12A. Tissue samples were also taken as indicated in FIG. 12A.Gastrocnemius (gastroc) muscle tissues were harvested and snap-frozen inliquid nitrogen. mRNA knockdown in target tissue was determined using acomparative qPCR assay as described in the methods section. Total RNAwas extracted from the tissue, reverse transcribed and mRNA levels werequantified using TaqMan qPCR, using the appropriately designed primersand probes. PPIB (housekeeping gene) was used as an internal RNA loadingcontrol, results were calculated by the comparative Ct method, where thedifference between the target gene Ct value and the PPIB Ct value (ΔCt)is calculated and then further normalized relative to the PBS controlgroup by taking a second difference (ΔΔCt). Quantitation of tissue siRNAconcentrations was determined using a stem-loop qPCR assay as describedin the methods section. The antisense strand of the siRNA was reversetranscribed using a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves.

Intracellular RISC loading was determined as described in Example 2.

Results

Quantifiable levels of siRNA accumulated in gastroc and heart tissue,see FIG. 12B, after a single intravenous dose of the antibody and FabsiRNA conjugates. Robust MSTN mRNA downregulation was observed ingastroc muscle, when the ASC was targeted with either theanti-transferrin receptor antibody or Fab see FIG. 12B and FIG. 12C.Much higher concentrations of siRNA were delivered to heart tissue, butthis did not result in robust myostatin mRNA downregulation, see FIG.12B. Compared to the cholesterol siRNA conjugate, much lower doses ofthe ASCs were required to achieve equivalent mRNA downregulation. Theamount of RISC loading of the MSTN siRNA guide strand correlated withdownregulation of the mRNA, see FIG. 12D.

Conclusions

In this example it was demonstrated that accumulation of siRNA ingastrocnemius muscle tissue after a single dose of an anti-transferrinantibody and Fab targeted siRNA conjugates. In Gastroc muscle, siRNAmediated MSTN mRNA downregulation with the DAR1 anti-transferrinantibody or Fab conjugates was observed. Mouse gastroc muscle expressestransferrin receptor and the conjugate have a mouse specificanti-transferrin antibody or Fab to target the payload, resulting inaccumulation of the conjugates in gastroc muscle and loading into theRISC complex. Receptor mediate uptake resulted in siRNA mediated MSTNmRNA downregulation.

Example 10: 2017-PK-304-WT—PK with MSTN Phenotype mAb Vs Chol siRNADesign and Synthesis

MSTN: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against mouse MSTN. The sequence (5′to 3′) of the guide/antisense strand was UUAUUAUUUGUUCUUUGCCUU (SEQ IDNO: 14226). Base, sugar and phosphate modifications that are welldescribed in the field of RNAi were used to optimize the potency of theduplex and reduce immunogenicity. All siRNA single strands were fullyassembled on solid phase using standard phospharamidite chemistry andpurified over HPLC. Purified single strands were duplexed to get thedouble stranded siRNA. The passenger strand contained two conjugationhandles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Bothconjugation handles were connected to siRNA passenger strand viaphosphorothioate-inverted abasic-phosphorothioate linker. Because thefree thiol was not being used for conjugation, it was end capped withN-ethylmaleimide.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 14228). The same base, sugar and phosphate modifications that wereused for the active MSTN siRNA duplex were used in the negative controlsiRNA. All siRNA single strands were fully assembled on solid phaseusing standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker. Because the free thiol was not beingused for conjugation, it was end capped with N-ethylmaleimide.

ASC Synthesis and Characterization

The CD71 mAb-siRNA DAR1 and DAR2 conjugates were made and characterizedas described in example 3. Groups 5-12 were made through cysteineconjugation, a SMCC linker, the free thiol was end capped with NEM usingarchitecture 1. Groups 13-16 were made through cysteine conjugation, aBisMal linker, the free thiol was end capped with NEM using architecture3. Conjugates were characterized chromatographically as described inTable 10.

TABLE 10 HPLC retention time (RT) in minutes RT, SAX RT, SEC GroupsConjugate Method-2 Method-1  5-8 TfR-mAb-MSTN; DAR1 9.5 7.9  9-12TfR-mAb-MSTN; DAR2 10.3 7.6 13-16 TfR-mAb-scramble; DAR1 9.1 7.3

In Vivo Study Design

The conjugates were assessed for their ability to mediate mRNAdownregulation of myostatin (MSTN) in skeletal muscle in vivo in wildtype CD-1 mice. Mice were dosed via intravenous (iv) injection with PBSvehicle control and the indicated ASCs at the doses indicated in FIG.13A. Tissue samples were also taken as indicated in FIG. 13A.Gastrocnemius (gastroc) muscle tissues were harvested and snap-frozen inliquid nitrogen. mRNA knockdown in target tissue was determined using acomparative qPCR assay as described in the methods section. Total RNAwas extracted from the tissue, reverse transcribed and mRNA levels werequantified using TaqMan qPCR, using the appropriately designed primersand probes. PPIB (housekeeping gene) was used as an internal RNA loadingcontrol, results were calculated by the comparative Ct method, where thedifference between the target gene Ct value and the PPIB Ct value (ΔCt)is calculated and then further normalized relative to the PBS controlgroup by taking a second difference (ΔΔCt). Quantitation of tissue siRNAconcentrations was determined using a stem-loop qPCR assay as describedin the methods section. The antisense strand of the siRNA was reversetranscribed using a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves.

Intracellular RISC loading was determined as described in Example 2.Plasma MSTN protein levels were measured by ELISA as described inExample 2.

Changes in leg muscle area were determined: the leg-to-be measured wereshaved and a line was drawn using indelible ink to mark region ofmeasurement. Mice were restrained in a small decapicone bag. Digitalcalipers were used to take one measurement on the sagittal plane andanother on the coronal plane. The procedure was repeated twice per week.

Results

Quantifiable levels of siRNA accumulated in gastrocnemius, triceps,quadriceps (Quad), and heart tissues, see FIG. 13D, after a singleintravenous dose of the antibody siRNA conjugates at 3 mg/kg. MSTN mRNAdownregulation was observed in gastrocnemius, quadriceps, and tricepswith the DAR1 and DAR2 conjugates but not in heart tissue, see FIG. 13B.MSTN mRNA downregulation resulted in a reduction in the plasmaconcentration of MSTN protein, as measured by ELISA, see FIG. 13C. Theamount of RISC loading of the MSTN siRNA guide strand correlated withdownregulation of the mRNA, see FIG. 13E. Downregulation of MSTNresulted in statistically significant increases in muscle size, see FIG.13F and FIG. 13G.

Conclusions

In this example it was demonstrated that accumulation of siRNA ingastrocnemius, quadriceps, and triceps muscle tissues after a singledose of anti-transferrin antibody siRNA conjugates, DAR1 and DAR2. Inall three tissues, measurable siRNA mediated MSTN mRNA downregulationwith the DAR1 and DAR2 anti-transferrin antibody conjugates wasobserved. mRNA downregulation correlated with a reduced level of plasmaMSTN protein and RISC loading of the siRNA guide strand. All threemuscle tissues expressed transferrin receptor and the conjugate has amouse specific anti-transferrin antibody to target the siRNA, resultingin accumulation of the conjugates in muscle. Receptor mediate uptakeresulted in siRNA mediated knockdown of the MSTN gene.

Example 11: 2017-PK-355-WT Multiple siRNA Dosing siRNA Design andSynthesis

HPRT: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against mouse MSTN. The sequence (5′to 3′) of the guide/antisense strand was UUAAAAUCUACAGUCAUAGUU (SEQ IDNO: 14227). Base, sugar and phosphate modifications that are welldescribed in the field of RNAi were used to optimize the potency of theduplex and reduce immunogenicity. All siRNA single strands were fullyassembled on solid phase using standard phospharamidite chemistry andpurified over HPLC. Purified single strands were duplexed to get thedouble stranded siRNA. The passenger strand contained a singleconjugation handles, a C6-NH₂ at the 5′, which was connected to siRNApassenger strand via phosphorothioate-inverted abasic-phosphorothioatelinker.

SSB: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotideoverhangs was designed against mouse MSTN. The sequence (5′ to 3′) ofthe guide/antisense strand was UUACAUUAAAGUCUGUUGUUU (SEQ ID NO: 14229).Base, sugar and phosphate modifications that are well described in thefield of RNAi were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained a single conjugation handles, a C6-NH₂ atthe 5′, which was connected to siRNA passenger strand viaphosphorothioate-inverted abasic-phosphorothioate linker.

ASC Synthesis and Characterization

The CD71 mAb-siRNA conjugates were made and characterized as describedin Example 3.

Groups 1-4 and 5-8 were made through cysteine conjugation, a BisMallinker, no 3′ conjugation handle on the passenger strand usingarchitecture 3. Groups 13-16 were made through cysteine conjugation, aBisMal linker, no 3′ conjugation handle on the passenger strand, butwere DAR2 conjugates made with a mixture of HPRT and SSB siRNAs usingarchitecture 4. Conjugates were characterized chromatographically asdescribed in Table 11.

TABLE 11 HPLC retention time (RT) in minutes Conjugate RT, SAX RT, SECMethod-2 Method-1 TfR-mAb-HPRT; DAR1 9.0 12.5 (0.5 ml flow rate,   25min run) TfR-mAb-SSB; DAR1 9.4 No Data TfR-mAb-HPRT/SSB (1:1) DAR2 10.09No Data

In Vivo Study Design

The conjugates were assessed for their ability to mediate mRNAdownregulation of two house keeper genes (HPRT and SSB) in skeletalmuscle in vivo in wild type CD-1 mice. Mice were dosed via intravenous(iv) injection with PBS vehicle control and the indicated ASCs at thedoses indicated in FIG. 14A. Tissue samples were also taken as indicatedin FIG. 14A. Gastrocnemius (gastroc) muscle tissues were harvested andsnap-frozen in liquid nitrogen. mRNA knockdown in target tissue wasdetermined using a comparative qPCR assay as described in the methodssection. Total RNA was extracted from the tissue, reverse transcribedand mRNA levels were quantified using TaqMan qPCR, using theappropriately designed primers and probes. PPIB (housekeeping gene) wasused as an internal RNA loading control, results were calculated by thecomparative Ct method, where the difference between the target gene Ctvalue and the PPIB Ct value (ΔCt) is calculated and then furthernormalized relative to the PBS control group by taking a seconddifference (ΔΔCt). Quantitation of tissue siRNA concentrations wasdetermined using a stem-loop qPCR assay as described in the methodssection. The antisense strand of the siRNA was reverse transcribed usinga TaqMan MicroRNA reverse transcription kit using a sequence-specificstem-loop RT primer. The cDNA from the RT step was then utilized forreal-time PCR and Ct values were transformed into plasma or tissueconcentrations using the linear equations derived from the standardcurves.

The RISC loading assay was conducted as described in Example 2.

Results

After a single intravenous dose of the antibody siRNA conjugates at theindicated doses, mRNA downregulation was observed in gastroc and hearttissue, see FIG. 14B-FIG. 14D. Co-administration of a mixture of twoASC's, targeting two different genes (HPRT and SSB) resulted inefficient mRNA downregulation of both targets in gastroc and hearttissue. In addition, administration of a DAR2 conjugate synthesizedusing a 1:1 mixture of the two different siRNAs (HPRT and SSB) alsoresulted in efficient mRNA down regulation of both targets in gastrocand heart tissue. All approaches to delivery resulted in measurableamounts of siRNA accumulating in gatroc tissue, see FIG. 14F.

Conclusions

In this example, it was demonstrated that accumulation of siRNA ingastroc and heart tissue after a single dose of anti-transferrinantibody siRNA conjugates. Two genes were downregulated byco-administration of two ASC produced with the same anti-transferrinantibody but conjugated to two different siRNAs (HPRT and SSB). Inaddition, two genes were downregulated by an anti-transferrin mAb DAR2conjugate synthesized using a 1:1 mixture of two different siRNAs (HPRTand SSB). In some instances, simultaneous downregulation of more thanone gene is useful in muscle atrophy.

Example 12: 2017-PK-380-WT Activity of Atrogin-1 siRNAs In Vivo (DoseResponse) siRNA Design and Synthesis

Atrogin-1 siRNAs: 4 different 21mer duplexes with 19 bases ofcomplementarity and 3′ dinucleotide overhangs were designed againstAtrogin-1, see Example 4 for details of the sequence. Base, sugar andphosphate modifications that are well described in the field of RNAiwere used to optimize the potency of the duplex and reduceimmunogenicity. The same design was used for all four siRNAs. All siRNAsingle strands were fully assembled on solid phase using standardphospharamidite chemistry and purified over HPLC. Purified singlestrands were duplexed to get the double stranded siRNA. The passengerstrand contained two conjugation handles, a C6-NH₂ at the 5′ end and aC6-SH at the 3′ end. Both conjugation handles were connected to siRNApassenger strand via phosphorothioate-inverted abasic-phosphorothioatelinker. Because the free thiol was not being used for conjugation, itwas end capped with N-ethylmaleimide.

ASC Synthesis and Characterization

The CD71 mAb-siRNA conjugates were made and characterized as describedin Example 3.

Groups 1-16 were made through cysteine conjugation, a BisMal linker, thefree thiol was end capped with NEM using architecture 3. Conjugates werecharacterized chromatographically as described Table 12.

TABLE 12 HPLC retention time (RT) in minutes RT, SAX RT, SEC GroupsConjugate Method-2 Method-1  1-4 mTfR1(Cys)-BisMal-N-mAtrogin#1179; DAR19.2 7.7  5-8 mTfR1(Cys)-BisMal-N-mAtrogin#1504; DAR1 9.3 7.8  9-12mTfR1(Cys)-BisMal-N-mAtrogin#631; DAR1 9.3 7.8 13-16mTfR1(Cys)-BisMal-N-mAtrogin#586; DAR1 9.2 7.8

In Vivo Study Design

The conjugates were assessed for their ability to mediate mRNAdownregulation of Atrogin-1 in skeletal muscle in vivo in wild type CD-1mice. Mice were dosed via intravenous (iv) injection with PBS vehiclecontrol and the indicated ASCs at the doses indicated in FIG. 15A.Tissue samples were taken as indicated in FIG. 15A. Gastrocnemius(gastroc) muscle tissues were harvested and snap-frozen in liquidnitrogen. mRNA knockdown in target tissue was determined using acomparative qPCR assay as described in the methods section. Total RNAwas extracted from the tissue, reverse transcribed and mRNA levels werequantified using TaqMan qPCR, using the appropriately designed primersand probes. PPIB (housekeeping gene) was used as an internal RNA loadingcontrol, results were calculated by the comparative Ct method, where thedifference between the target gene Ct value and the PPIB Ct value (ΔCt)is calculated and then further normalized relative to the PBS controlgroup by taking a second difference (ΔΔCt).

Results

After a single intravenous dose of the antibody siRNA conjugates at theindicated doses, up to 80% atrogin-1 mRNA downregulation was observed ingastroc muscle and up to 50% in heart tissue, see FIG. 15B and FIG. 15C.

Conclusions

As illustrated in this example, antibody siRNA conjugates differentiallydownregulate Atrogin-1 in muscle and heart.

Example 13: 2017-PK-383-WT Activity of MuRF1 siRNA In Vivo (DoseResponse) siRNA Design and Synthesis

MuRF1 siRNAs: 4 different 21mer duplexes with 19 bases ofcomplementarity and 3′ dinucleotide overhangs were designed againstAtrogin-1, see Example 5 for details of the sequence. Base, sugar andphosphate modifications that are well described in the field of RNAiwere used to optimize the potency of the duplex and reduceimmunogenicity. The same design was used for all four siRNAs. All siRNAsingle strands were fully assembled on solid phase using standardphospharamidite chemistry and purified over HPLC. Purified singlestrands were duplexed to get the double stranded siRNA. The passengerstrand contained two conjugation handles, a C6-NH₂ at the 5′ end and aC6-SH at the 3′ end. Both conjugation handles were connected to siRNApassenger strand via phosphorothioate-inverted abasic-phosphorothioatelinker. Because the free thiol was not being used for conjugation, itwas end capped with N-ethylmaleimide.

ASC Synthesis and Characterization

The CD71 mAb-siRNA conjugates were made and characterized as describedin Example 3.

Groups 1-16 were made through cysteine conjugation, a BisMal linker, thefree thiol was end capped with NEM using architecture 3. Conjugates werecharacterized chromatographically as described Table 13.

TABLE 13 HPLC retention time (RT) in minutes RT, SAX RT, SEC GroupsConjugate Method-2 Method-1  1-4 mTfR1(Cys)-BisMal-N-MuRF#651-S-NEM;DAR1 9.2 7.8  5-8 mTfR1(Cys)-BisMal-N-MuRF#1387-S-NEM; DAR1 9.3 7.8 9-13 mTfR1(Cys)-BisMal-N-MuRF#1454-S-NEM; DAR1 9.1 7.8 14-18mTfR1(Cys)-BisMal-N-MuRF#1660-S-NEM; DAR1 9.1 7.8

In Vivo Study Design

The conjugates were assessed for their ability to mediate mRNAdownregulation of MuRF-1 in skeletal and heart muscle in vivo in wildtype CD-1 mice. Mice were dosed via intravenous (iv) injection with PBSvehicle control and the indicated ASCs at the doses indicated in FIG.16A. Tissue samples were taken as indicated in FIG. 16A. Gastrocnemius(gastroc) muscle tissues were harvested and snap-frozen in liquidnitrogen. mRNA knockdown in target tissue was determined using acomparative qPCR assay as described in the methods section. Total RNAwas extracted from the tissue, reverse transcribed and mRNA levels werequantified using TaqMan qPCR, using the appropriately designed primersand probes. PPIB (housekeeping gene) was used as an internal RNA loadingcontrol, results were calculated by the comparative Ct method, where thedifference between the target gene Ct value and the PPIB Ct value (ΔCt)is calculated and then further normalized relative to the PBS controlgroup by taking a second difference (ΔΔCt).

Results

After a single intravenous dose of the antibody siRNA conjugates at theindicated doses, MuRF1 mRNA in gastroc muscle was downregulated to up to70% and up to 50% in heart tissue, see FIG. 16B and FIG. 16C.

Conclusions

As illustrated in this example, antibody siRNA conjugates differentiallydownregulate MuRF1 in muscle and heart.

Example 14

Table 14 illustrates exemplary siRNA (or atrogene) targets to regulatemuscle atrophy. In some instances, a polynucleic acid moleculehybridizes to a target region of an atrogene described in Table 14

Function Gene Name Protein Degradation FBXO32 Atrogin-1 Trim63 MuRF1TRAF6 TNF receptor-associated factor 6 USP14 Ubiquitin specific protease14 CTSL2 Cathepsin L2 Transcription Foxo1 Forkhead box 01 Foxo3 Forkheadbox 03 TGIF TG interacting factor MYOG myogenin HDAC2 Histonedeacetylase 2 HDAC3 Histone deacetylase 3 Stress Response MT1LMetallothionein 1L MT1B Metallothionein 1B

Example 15: Sequences

23-mer target sequences within one DMPK transcript variant(NM_001288766) are assigned with SEQ ID NOs: 703-3406. The set of 23-mertarget sequences for this transcript variant was generated by walkingdown the length of the transcript one base at a time, and a similar setof target sequences could be generated for the other DMPK transcriptvariants using the same procedure. One common siRNA structure that canbe used to target these sites in the DMPK transcript is a 19-mer fullycomplimentary duplex with 2 overhanging (not base-paired) nucleotides onthe 3′ end of each strand. Thus, adding the 19-mer with both of the 2nucleotide overhangs results in a total of 23 bases for the target site.Since the overhangs can be comprised of a sequence reflecting that ofthe target transcript or other nucleotides (for example a non-relateddinucleotide sequence such as “UU”), the 19-mer fully complimentarysequence can be used to describe the siRNA for each 23-mer target site.

19-mer sense and antisense sequences for siRNA duplexes targeting eachsite within the DMPK transcript are assigned with SEQ ID NOs: 3407-8814(with the first sense and antisense pairing as SEQ ID NO: 3407 and SEQID NO: 6111). SEQ ID NOs: 3407-6110 illustrate the sense strand. SEQ IDNOs: 6111-8814 illustrate the antisense strand. The DMPK transcriptvariant NM_001288766 has been used for illustration but a similar set ofsiRNA duplexes can be generated by walking through the other DMPKtranscript variants. When the antisense strand of the siRNA loads intoAgo2, the first base associates within the Ago2 binding pocket while theother bases (starting at position 2 of the antisense strand) aredisplayed for complimentary mRNA binding. Since “U” is thethermodynamically preferred first base for binding to Ago2 and does notbind the target mRNA, all of the antisense sequences can have “U”substituted into the first base without affecting the targetcomplementarity and specificity. Correspondingly, the last base of thesense strand 19-mer (position 19) is switched to “A” to ensure basepairing with the “U” at the first position of the antisense strand.

SEQ ID NOs: 8815-11518 are similar to SEQ ID NOs: 3407-6110 except thelast position of the 19-mer sense strand substituted with base “A”.

SEQ ID NOs: 11519-14222 are similar to SEQ ID NOs: 6111-8814 except thefirst position of the 19-mer antisense strand substituted with base “U”.

SEQ ID NO: 8815 and SEQ ID NO: 11519 for the first respective sense andantisense pairing.

Example 16: Initial Screening of a Selected Set of DMPK siRNAs for InVitro Activity

The initial set of DMPK siRNAs from SEQ ID NOs: 8815-14222 was narroweddown to a list of 81 siRNA sequences using a bioinformatic analysisaimed at selecting the sequences with the highest probability ofon-target activity and the lowest probability of off-target activity.The bioinformatic methods for selecting active and specific siRNAs arewell described in the field of RNAi and a person skilled in the artswould be able to generate a similar list of DMPK siRNA sequences againstany of the other DMPK transcript variants. The DMPK siRNAs in the set of81 sequences were synthesized on small scale using standard solid phasesynthesis methods that are described in the oligonucleotide synthesisliterature. Both unmodified and chemically modified siRNAs are known toproduce effective knockdown following in vitro transfection. The DMPKsiRNA sequences were synthesized using base, sugar and phosphatemodifications that are described in the field of RNAi to optimize thepotency of the duplex and reduce immunogenicity. Two human cell lineswere used to assess the in vitro activity of the DMPK siRNAs: first,SJCRH30 human rhabdomyosarcoma cell line (ATCC® CRL-2061™); and second,Myotonic Dystrophy Type 1 (DM1) patient-derived immortalized humanskeletal myoblasts. For the initial screening of the DMPK siRNA library,each DMPK siRNA was transfected into SJCRH30 cells at 1 nM and 0.01 nMfinal concentration, as well as into DM1 myoblasts at 10 nM and 1 nMfinal concentration. The siRNAs were formulated with transfectionreagent Lipofectamine RNAiMAX (Life Technologies) according to themanufacturer's “forward transfection” instructions. Cells were plated 24h prior to transfection in triplicate on 96-well tissue culture plates,with 8500 cells per well for SJCRH30 and 4000 cells per well for DM1myoblasts. At 48 h (SJCRH30) or 72 h (DM1 myoblasts) post-transfectioncells were washed with PBS and harvested with TRIzol® reagent (LifeTechnologies). RNA was isolated using the Direct-zol-96 RNA Kit (ZymoResearch) according to the manufacturer's instructions. 10 μl of RNA wasreverse transcribed to cDNA using the High Capacity cDNA ReverseTranscription Kit (Applied Biosystems) according to the manufacturer'sinstructions. cDNA samples were evaluated by qPCR with DMPK-specific andPPIB-specific TaqMan human gene expression probes (Thermo Fisher) usingTaqMan® Fast Advanced Master Mix (Applied Biosystems). DMPK values werenormalized within each sample to PPIB gene expression. Thequantification of DMPK downregulation was performed using the standard2^(−ΔΔCt) method. All experiments were performed in triplicate, withTable 15A and Table 15B) presenting the mean values of the triplicates.

TABLE 15A sense antisense strand strand sequence sequence (5′-3′)(5′-3′) Passenger SEQ Guide SEQ Strand ID Strand ID ID#¹ (PS) NO: (GS)NO: 385 GCUUA 9199 UAGUC 11903 AGGAG GGACC GUCCG UCCUU ACUA AAGC 443GGGGC 9257 UUACC 11961 GUUCA UCGCU GCGAG GAACG GUAA CCCC 444 GGGCG 9258UCUAC 11962 UUCAG CUCGC CGAGG UGAAC UAGA GCCC 445 GGCGU 9259 UGCUA 11963UCAGC CCUCG GAGGU CUGAA AGCA CGCC 533 AGGGG 9347 UGCAC 12051 CGAGG GACACUGUCG CUCGC UGCA CCCU 534 GGGGC 9348 UAGCA 12052 GAGGU CGACA GUCGU CCUCGGCUA CCCC 535 GGGCG 9349 UAAGC 12053 AGGUG ACGAC UCGUG ACCUC CUUA GCCC539 GAGGU 9353 UACGG 12057 GUCGU AAGCA GCUUC CGACA CGUA CCUC 540 AGGUG9354 UCACG 12058 UCGUG GAAGC CUUCC ACGAC GUGA ACCU 541 GGUGU 9355 UUCAC12059 CGUGC GGAAG UUCCG CACGA UGAA CACC 543 UGUCG 9357 UCCUC 12061 UGCUUACGGA CCGUG AGCAC AGGA GACA 544 GUCGU 9358 UUCCU 12062 GCUUC CACGG CGUGAAAGCA GGAA CGAC 576 UGAAU 9390 UACCG 12094 GGGGA CCGGU CCGGC CCCCA GGUAUUCA 577 GAAUG 9391 UCACC 12095 GGGAC GCCGG CGGCG UCCCC GUGA AUUC 581GGGGA 9395 UGAUC 12099 CCGGC CACCG GGUGG CCGGU AUCA CCCC 583 GGACC 9397UGUGA 12101 GGCGG UCCAC UGGAU CGCCG CACA GUCC 584 GACCG 9398 UCGUG 12102GCGGU AUCCA GGAUC CCGCC ACGA GGUC 690 AGUUU 9504 UGAAU 12208 GGGGA CCGCUGCGGA CCCCA UUCA AACU 716 AUGGC 9530 UCAGG 12234 GCGCU UAGAA UCUAC GCGCGCUGA CCAU 717 UGGCG 9531 UCCAG 12235 CGCUU GUAGA CUACC AGCGC UGGA GCCA785 AGGGA 9599 UGUCG 12303 CAUCA GGUUU AACCC GAUGU GACA CCCU 786 GGGAC9600 UUGUC 12304 AUCAA GGGUU ACCCG UGAUG ACAA UCCC 789 ACAUC 9603 UUGUU12307 AAACC GUCGG CGACA GUUUG ACAA AUGU 1026 GGCAG 9840 UCGUA 12544ACGCC GAAGG CUUCU GCGUC ACGA XJGCC 1027 GCAGA 9841 UGCGU 12545 CGCCCAGAAG UUCUA GGCGU CGCA CUGC 1028 CAGAC 9842 UCGCG 12546 GCCCU UAGAAUCUAC GGGCG GCGA UCUG 1029 AGACG 9843 UCCGC 12547 CCCUU GUAGA CUACGAGGGC CGGA GUCU 1037 UUCUA 9851 UCGUG 12555 CGCGG GAAUC AUUCC CGCGU ACGAAGAA 1039 CUACG 9853 UGCCG 12557 CGGAU UGGAA UCCAC UCCGC GGCA GUAG 1041ACGCG 9855 UCCGC 12559 GAUUC CGUGG CACGG AAUCC CGGA GCGU 1043 GCGGA 9857UCUCC 12561 UUCCA GCCGU CGGCG GGAAU GAGA CCGC 1044 CGGAU 9858 UUCUC12562 UCCAC CGCCG GGCGG UGGAA AGAA UCCG 1047 AUUCC 9861 UAGGU 12565ACGGC CUCCG GGAGA CCGUG CCUA GAAU 1071 AGAUC 9885 UCCUU 12589 GUCCAGUAGU CUACA GGACG AGGA AUCU 1073 AUCGU 9887 UCUCC 12591 CCACU UUGUAACAAG GUGGA GAGA CGAU 1262 CCCUU 10076 UGAAA 12780 UACAC UCCGG CGGAUUGUAA UUCA AGGG 1263 CCUUU 10077 UCGAA 12781 ACACC AUCCG GGAUU GUGUAUCGA AAGG 1264 CUUUA 10078 UUCGA 12782 CACCG AAUCC GAUUU GGUGU CGAA AAAG1265 UUUAC 10079 UUUCG 12783 ACCGG AAAUC AUUUC CGGUG GAAA UAAA 1267UACAC 10081 UCCUU 12785 CGGAU CGAAA UUCGA UCCGG AGGA UGUA 1268 ACACC10082 UACCU 12786 GGAUU UCGAA UCGAA AUCCG GGUA GUGU 1269 CACCG 10083UCACC 12787 GAUUU UUCGA CGAAG AAUCC GUGA GGUG 1274 GAUUU 10088 UGGUG12792 CGAAG GCACC GUGCC UUCGA ACCA AAUC 1276 UUUCG 10090 UUCGG 12794AAGGU UGGCA GCCAC CCUUC CGAA GAAA 1283 GGUGC 10097 UGCAU 12801 CACCGGUGUC ACACA GGUGG UGCA CACC 1297 AUGCA 10111 UACCA 12815 ACUUC AGUCGGACUU AAGUU GGUA GCAU 1342 ACUGU 10156 UUCCC 12860 CGGAC GAAUG AUUCGUCCGA GGAA CAGU 1343 CUGUC 10157 UUUCC 12861 GGACA CGAAU UUCGG GUCCGGAAA ACAG 1344 UGUCG 10158 UCUUC 12862 GACAU CCGAA UCGGG UGUCC AAGA GACA1346 UCGGA 10160 UACCU 12864 CAUUC UCCCG GGGAA AAUGU GGUA CCGA 1825UGCUC 10639 UCAAC 13343 CUGUU GGCGA CGCCG ACAGG UUGA AGCA 1886 CCACG10700 UGUGA 13404 CCGGC GUUGG CAACU CCGGC CACA GUGG 1890 GCCGG 10704UUGCG 13408 CCAAC GUGAG UCACC UUGGC GCAA CGGC 1898 ACUCA 10712 UCGCC13416 CCGCA AGACU GUCUG GCGGU GCGA GAGU 1945 CCCUA 10759 UUCGA 13463GAACU AGACA GUCUU GUUCU CGAA AGGG 1960 CGACU 10774 UAACG 13478 CCGGGGGGCC GCCCC CCGGA GUUA GUCG 2126 GCCGG 10940 UCGAG 13644 CGAAC CCCCGGGGGC UUCGC UCGA CGGC 2127 CCGGC 10941 UUCGA 13645 GAACG GCCCC GGGCUGUUCG CGAA CCGG 2149 UCCUU 10963 UCAUU 13667 GUAGC CCCGG CGGGA CUACAAUGA AGGA 2150 CCUUG 10964 UGCAU 13668 UAGCC UCCCG GGGAA GCUAC UGCA AAGG2268 CCCUG 11082 UUGCC 13786 ACGUG CAUCC GAUGG ACGUC GCAA AGGG 2272GACGU 11086 UAGUU 13790 GGAUG UGCCC GGCAA AUCCA ACUA CGUC 2528 GCUUC11342 UUAUC 14046 GGCGG CAAAC UUUGG CGCCG AUAA AAGC 2529 CUUCG 11343UAUAU 14047 GCGGU CCAAA UUGGA CCGCC UAUA GAAG 2530 UUCGG 11344 UAAUA14048 CGGUU UCCAA UGGAU ACCGC AUUA CGAA 2531 UCGGC 11345 UAAAU 14049GGUUU AUCCA GGAUA AACCG UUUA CCGA 2532 CGGCG 11346 UUAAA 14050 GUUUGUAUCC GAUAU AAACC UUAA GCCG 2554 CCUCG 11368 UGCGA 14072 UCCUC GUCGGCGACU AGGAC CGCA GAGG 2558 GUCCU 11372 UGUCA 14076 CCGAC GCGAG UCGCUUCGGA GACA GGAC 2600 CAAUC 11414 UCAUC 14118 CACGU CAAAA UUUGG CGUGGAUGA AUUG 2628 CCGAC 11442 UAAUA 14146 AUUCC CCGAG UCGGU GAAUG AUUA UCGG2629 CGACA 11443 UAAAU 14147 UUCCU ACCGA CGGUA GGAAU UUUA GUCG 2631ACAUU 11445 UAUAA 14149 CCUCG AUACC GUAUU GAGGA UAUA AUGU 2636 CCUCG11450 UAGAC 14154 GUAUU AAUAA UAUUG AUACC UCUA GAGG 2639 CGGUA 11453UGACA 14157 UUUAU GACAA UGUCU UAAAU GUCA ACCG 2675 CCCCG 11489 UUAUU14193 ACCCU CGCGA CGCGA GGGUC AUAA GGGG 2676 CCCGA 11490 UUUAU 14194CCCUC UCGCG GCGAA AGGGU UAAA CGGG 2679 GACCC 11493 UCUUU 14197 UCGCGUAUUC AAUAA GCGAG AAGA GGUC 2680 ACCCU 11494 UCCUU 14198 CGCGA UUAUUAUAAA CGCGA AGGA GGGU 2681 CCCUC 11495 UGCCU 14199 GCGAA UUUAU UAAAAUCGCG GGCA AGGG 2682 CCUCG 11496 UGGCC 14200 CGAAU UUUUA AAAAG UUCGCGCCA GAGG Neg. n/a n/a n/a n/a Control ¹19mer position in NM_001288766.1

TABLE 15B ID #¹ qPCR² qPCR³ qPCR⁴ qPCR⁵ 385 150.8 153.8 64.1 142.5 443112.7 95.8 56.7 127.8 444 76.5 66.2 36.7 113.6 445 61.4 107.7 29.4 110.8533 168.8 119.8 85.7 118.1 534 91.4 44.8 26.7 94.2 535 101.0 65.9 33.1109.9 539 81.7 70.2 34.1 102.4 540 68.3 56.8 40.0 114.6 541 112.1 107.373.8 120.6 543 42.6 59.9 41.9 117.8 544 42.4 107.5 66.9 154.7 576 107.4119.0 85.0 127.5 577 101.6 90.1 72.1 106.6 581 199.3 97.7 69.9 103.5 58366.6 77.5 66.4 100.3 584 26.3 37.3 31.0 88.3 690 163.6 84.1 58.0 92.7716 29.0 39.6 29.4 86.0 717 44.4 45.7 52.8 102.5 785 79.9 93.2 71.3101.0 786 85.5 63.8 54.3 92.2 789 45.4 51.3 43.8 96.9 1026 55.6 77.332.0 110.4 1027 98.9 94.7 35.2 108.3 1028 132.1 104.9 27.3 87.3 102962.2 95.5 45.9 94.2 1037 68.2 80.2 65.3 97.0 1039 42.3 79.3 53.6 97.01041 67.2 64.4 73.2 98.6 1043 342.8 86.6 61.5 96.5 1044 109.5 84.8 42.794.0 1047 101.3 72.1 35.2 90.7 1071 88.5 99.6 91.6 101.3 1073 134.3 63.036.3 93.6 1262 36.5 59.6 27.3 117.5 1263 47.6 79.7 33.9 104.3 1264 64.254.5 43.7 95.4 1265 19.8 57.6 30.9 91.6 1267 61.3 85.9 73.4 97.1 126832.0 28.3 38.0 92.3 1269 42.6 49.1 42.4 96.7 1274 63.6 55.4 78.0 98.51276 52.2 36.9 35.2 82.5 1283 35.2 62.8 56.6 95.9 1297 20.3 55.7 32.291.0 1342 44.6 46.7 41.5 94.5 1343 65.8 80.0 56.1 119.2 1344 30.9 63.751.7 116.7 1346 133.8 102.9 98.0 104.0 1825 54.1 69.2 28.6 86.7 1886786.9 282.0 130.5 98.4 1890 28.8 30.3 51.5 94.4 1898 125.5 57.5 67.797.6 1945 23.5 22.6 21.8 57.6 1960 28.4 33.7 35.7 87.9 2126 147.9 87.286.8 98.1 2127 46.5 51.9 52.7 96.2 2149 44.7 41.5 62.0 99.6 2150 110.489.1 63.4 114.1 2268 53.5 48.6 60.8 113.1 2272 56.5 54.7 46.9 92.5 252832.5 32.8 32.7 76.9 2529 19.6 25.8 21.4 59.5 2530 29.5 25.9 32.8 68.12531 22.2 31.6 25.4 64.3 2532 44.4 35.6 29.2 74.0 2554 13.7 22.6 26.860.9 2558 54.6 47.4 28.0 72.0 2600 205.4 209.6 n.d. n.d. 2628 12.6 28.520.1 56.2 2629 12.8 39.5 20.6 63.8 2631 97.4 68.6 39.7 104.4 2636 62.068.6 16.8 58.7 2639 33.2 46.1 22.2 81.0 2675 57.7 82.5 n.d. n.d. 267631.1 53.0 n.d. n.d. 2679 44.7 75.7 n.d. n.d. 2680 89.2 61.5 n.d. n.d.2681 19.0 28.6 n.d. n.d. 2682 98.2 61.8 n.d. n.d. Neg. Control 101.2100.6 101.1 106.4 ²DM1 myoblasts; 10 nM; % DMPK mRNA ³DM1 myoblasts; 1nM; % DMPK mRNA ⁴SJCRH30; 1 nM; % DMPK mRNA ⁵SJCRH30; 0.01 nM; % DMPKmRNA

Example 17: In Vitro Dose Response Curves for a Selected Set of DMPKsiRNAs

To further validate the activity of the DMPK siRNAs, many of thesequences that showed the best activity in the initial screen wereselected for a follow-up evaluation in dose response format. Once again,two human cell lines were used to assess the in vitro activity of theDMPK siRNAs: first, SJCRH30 human rhabdomyosarcoma cell line; andsecond, Myotonic Dystrophy Type 1 (DM1) patient-derived immortalizedhuman skeletal myoblasts. The selected siRNAs were transfected in a10-fold dose response at 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 nMfinal concentrations or in a 9-fold dose response at 50, 5.55556,0.617284, 0.068587, 0.007621, 0.000847, and 0.000094 nM finalconcentrations. The siRNAs were formulated with transfection reagentLipofectamine RNAiMAX (Life Technologies) according to themanufacturer's “forward transfection” instructions. Cells were plated 24h prior to transfection in triplicate on 96-well tissue culture plates,with 8500 cells per well for SJCRH30 and 4000 cells per well for DM1myoblasts. At 48 h (SJCRH30) or 72 h (DM1 myoblasts) post-transfectioncells were washed with PBS and harvested with TRIzol® reagent (LifeTechnologies). RNA was isolated using the Direct-zol-96 RNA Kit (ZymoResearch) according to the manufacturer's instructions. 10 μl of RNA wasreverse transcribed to cDNA using the High Capacity cDNA ReverseTranscription Kit (Applied Biosystems) according to the manufacturer'sinstructions. cDNA samples were evaluated by qPCR with DMPK-specific andPPIB-specific TaqMan human gene expression probes (Thermo Fisher) usingTaqMan® Fast Advanced Master Mix (Applied Biosystems). DMPK values werenormalized within each sample to PPIB gene expression. Thequantification of DMPK downregulation was performed using the standard2^(−ΔΔCt) method. All experiments were performed in triplicate, withTables 16A-B, 17A-B, and 18A-B presenting the mean values of thetriplicates as well as the calculated IC₅₀ values determined fromfitting curves to the dose-response data by non-linear regression.

TABLE 16A sense antisense strand strand sequence sequence (5′-3′)(5′-3′) Passenger SEQ Guide SEQ Strand ID Strand ID ID#¹ (PS) NO: (GS)NO: 535 GGGCG 9349 UAAGC 12053 AGGUG ACGAC UCGUG ACCUC CUUA GCCC 584GACCG 9398 UCGUG 12102 GCGGU AUCCA GGAUC CCGCC ACGA GGUC 716 AUGGC 9530UCAGG 12234 GCGCU UAGAA UCUAC GCGCG CUGA CCAU 1028 CAGAC 9842 UCGCG12546 GCCCU UAGAA UCUAC GGGCG GCGA UCUG 1276 UUUCG 10090 UUCGG 12794AAGGU UGGCA GCCAC CCUUC CGAA GAAA 1825 UGCUC 10639 UCAAC 13343 CUGUUGGCGA CGCCG ACAGG UUGA AGCA 1945 CCCUA 10759 UUCGA 13463 GAACU AGACAGUCUU GUUCU CGAA AGGG 2529 CUUCG 11343 UAUAU 14047 GCGGU CCAAA UUGGACCGCC UAUA GAAG 2558 GUCCU 11372 UGUCA 14076 CCGAC GCGAG UCGCU UCGGAGACA GGAC 2628 CCGAC 11442 UAAUA 14146 AUUCC CCGAG UCGGU GAAUG AUUA UCGG2636 CCUCG 11450 UAGAC 14154 GUAUU AAUAA UAUUG AUACC UCUA GAGG ¹19merposition in NM_001288766.1

TABLE 16B IC50 ID #¹ qPCR² qPCR³ qPCR⁴ qPCR⁵ qPCR⁶ qPCR⁷ qPCR⁸ (nM) 535111.9 105.4 106.3 82.4 36.7 29.5 35.7 0.165 584 90.5 90.2 84.7 67.8 38.025.8 28.3 0.190 716 88.9 85.2 81.9 62.0 32.6 19.3 20.3 0.181 1028 88.581.8 83.0 61.3 32.7 27.3 31.5 0.127 1276 87.0 85.0 84.0 66.1 40.5 34.036.4 0.150 1825 85.1 85.9 83.7 69.1 36.2 25.2 25.0 0.259 1945 85.0 81.774.4 44.9 22.9 17.7 17.2 0.070 2529 83.3 81.8 75.3 50.6 24.6 17.5 17.70.103 2558 84.3 81.1 74.3 45.4 23.4 13.3 11.8 0.088 2628 85.3 84.0 79.559.8 30.3 23.5 25.1 0.140 2636 86.3 86.9 74.3 44.0 19.8 12.4 13.0 0.070²SJCRH30; 0.0001 nM; % DMPK mRNA ³SJCRH30; 0.001 nM; % DMPK mRNA⁴SJCRH30; 0.01 nM; % DMPK mRNA ⁵SJCRH30; 0.1 nM; % DMPK mRNA ⁶SJCRH30; 1nM; % DMPK mRNA ⁷SJCRH30; 10 nM; % DMPK mRNA ⁸SJCRH30; 100 nM; % DMPKmRNA

TABLE 17A sense antisense strand strand sequence sequence (5′-3′)(5′-3′) Passenger SEQ Guide SEQ Strand ID Strand ID ID#¹ (PS) NO: (GS)NO: 2600 CAAUC 11414 UCAUC 14118 CACGU CAAAA UUUGG CGUGG AUGA AUUG 2636CCUCG 11450 UAGAC 14154 GUAUU AAUAA UAUUG AUACC UCUA GAGG 2675 CCCCG11489 UUAUU 14193 ACCCU CGCGA CGCGA GGGUC AUAA GGGG 2676 CCCGA 11490UUUAU 14194 CCCUC UCGCG GCGAA AGGGU UAAA CGGG 2679 GACCC 11493 UCUUU14197 UCGCG UAUUC AAUAA GCGAG AAGA GGUC 2680 ACCCU 11494 UCCUU 14198CGCGA UUAUU AUAAA CGCGA AGGA GGGU 2681 CCCUC 11495 UGCCU 14199 GCGAAUUUAU UAAAA UCGCG GGCA AGGG 2682 CCUCG 11496 UGGCC 14200 CGAAU UUUUAAAAAG UUCGC GCCA GAGG ¹19mer position in NM_001288766.1

TABLE 17B IC50 ID #¹ qPCR² qPCR³ qPCR⁴ qPCR⁵ qPCR⁶ qPCR⁷ (nM) 2600 107.5107.6 108.1 106.3 103.1 72.7 31.31 2636 81.1 81.1 74.0 47.2 25.7 11.50.073 2675 88.1 88.3 84.3 64.6 38.1 20.7 0.151 2676 88.9 78.9 84.4 72.744.9 35.6 0.204 2679 84.0 87.3 82.7 53.3 31.4 13.5 0.091 2680 87.4 85.385.1 68.5 44.5 39.6 0.110 2681 87.0 85.4 77.6 49.6 26.5 16.0 0.061 268282.4 83.9 77.1 50.8 27.3 31.1 0.047 ²SJCRH30. 0.000094 nM; % DMPK mRNA³SJCRH30. 0.000847 nM; % DMPK mRNA ⁴SJCRH30. 0.007621 nM; % DMPK mRNA⁵SJCRH30. 0.068587 nM; % DMPK mRNA ⁶SJCRH30. 0.617284 nM; % DMPK mRNA⁷SJCRH30. 5.55556 nM; % DMPK mRNA

TABLE 18A sense antisense strand strand sequence sequence (5′-3′)(5′-3′) Passenger SEQ Guide SEQ Strand ID Strand ID ID#¹ (PS) NO: (GS)NO: 584 GACCG 9398 UCGUG 12102 GCGGU AUCCA GGAUC CCGCC ACGA GGUC 716AUGGC 9530 UCAGG 12234 GCGCU UAGAA UCUAC GCGCG CUGA CCAU 1265 UUUAC10079 UUUCG 12783 ACCGG AAAUC AUUUC CGGUG GAAA UAAA 1297 AUGCA 10111UACCA 12815 ACUUC AGUCG GACUU AAGUU GGUA GCAU 1945 CCCUA 10759 UUCGA13463 GAACU AGACA GUCUU GUUCU CGAA AGGG 1960 CGACU 10774 UAACG 13478CCGGG GGGCC GCCCC CCGGA GUUA GUCG 2529 CUUCG 11343 UAUAU 14047 GCGGUCCAAA UUGGA CCGCC UAUA GAAG 2530 UUCGG 11344 UAAUA 14048 CGGUU UCCAAUGGAU ACCGC AUUA CGAA 2531 UCGGC 11345 UAAAU 14049 GGUUU AUCCA GGAUAAACCG UUUA CCGA 2554 CCUCG 11368 UGCGA 14072 UCCUC GUCGG CGACU AGGACCGCA GAGG 2628 CCGAC 11442 UAAUA 14146 AUUCC CCGAG UCGGU GAAUG AUUA UCGG2629 CGACA 11443 UAAAU 14147 UUCCU ACCGA CGGUA GGAAU UUUA GUCG 2681CCCUC 11495 UGCCU 14199 GCGAA UUUAU UAAAA UCGCG GGCA AGGG ¹19merposition in NM_001288766.1

TABLE 18B IC50 ID #1 qPCR² qPCR³ qPCR⁴ qPCR⁵ qPCR⁶ qPCR⁷ (nM) 584 90.877.0 97.7 71.9 45.0 29.7 0.228 716 96.5 82.5 77.0 64.6 43.3 33.9 0.0801265 68.5 80.9 68.0 57.1 37.5 25.7 0.146 1297 71.4 67.2 69.4 53.5 40.525.4 0.171 1945 71.8 62.3 41.7 29.8 22.4 15.3 0.006 1960 63.0 65.4 62.145.8 31.1 28.3 0.068 2529 63.5 58.7 49.2 31.1 22.9 21.9 0.017 2530 69.366.7 53.1 43.2 38.8 24.5 0.016 2531 69.9 72.4 57.3 40.2 35.4 25.6 0.0182554 68.2 70.1 51.2 43.0 32.1 17.3 0.043 2628 69.7 67.9 62.5 38.4 31.617.1 0.042 2629 72.1 65.6 69.0 42.1 34.4 13.7 0.078 2681 82.4 91.5 87.655.5 29.3 19.6 0.084 ²DM1 myoblasts; 0.000094 nM; % DMPK mRNA ³DM1myoblasts; 0.000847 nM; % DMPK mRNA ⁴DM1 myoblasts; 0.007621 nM; % DMPKmRNA ⁵DM1 myoblasts; 0.068587 nM; % DMPK mRNA ⁶DM1 myoblasts; 0.617284nM; % DMPK mRNA ⁷DM1 myoblasts; 5.55556 nM; % DMPK mRNA

Example 18: In Vitro Experiments to Determine Species Cross-Reactivityin Mouse

The selected siRNAs were transfected at 100, 10, 1, 0.1, 0.01, 0.001,and 0.0001 nM final concentrations into C2C12 mouse muscle myoblasts(ATCC® CRL-1772™). The siRNAs were formulated with transfection reagentLipofectamine RNAiMAX (Life Technologies) according to themanufacturer's “forward transfection” instructions. Cells were plated 24h prior to transfection in triplicate on 96-well tissue culture plates,with 4000 cells per well for C2C12 seeding. At 48 h post-transfectioncells were washed with PBS and harvested with TRIzol® reagent (LifeTechnologies). RNA was isolated using the Direct-zol-96 RNA Kit (ZymoResearch) according to the manufacturer's instructions. 10 μl of RNA wasreverse transcribed to cDNA using the High Capacity cDNA ReverseTranscription Kit (Applied Biosystems) according to the manufacturer'sinstructions. cDNA samples were evaluated by qPCR with DMPK-specific andPPIB-specific TaqMan mouse gene expression probes (Thermo Fisher) usingTaqMan® Fast Advanced Master Mix (Applied Biosystems). DMPK values werenormalized within each sample to PPIB gene expression. Thequantification of DMPK downregulation was performed using the standard2^(−ΔΔCt) method. All experiments were performed in triplicate, with theresults shown in FIG. 17. Four DMPK siRNAs (the numbers indicated in theFIG. 17 legend correspond to the ID # that is listed in Table 19 (Tables19A-19B)) were shown to effectively cross-react with mouse DMPK mRNA,producing robust mRNA knockdown in the mouse C2C12 myoblast cell line.Two of the siRNAs (ID #s 535 and 1028) were slightly less effective andonly produced approximately 70% maximum mRNA knockdown. Two of thesiRNAs (ID #s 2628 and 2636) were more effective and producedapproximately 90% maximum mRNA knockdown.

Example 19: In Vivo Experiments to Determine Species Cross-Reactivity inMouse

Animals

All animal studies were conducted following protocols in accordance withthe Institutional Animal Care and Use Committee (IACUC) at ExploraBioLabs, which adhere to the regulations outlined in the USDA AnimalWelfare Act as well as the “Guide for the Care and Use of LaboratoryAnimals” (National Research Council publication, 8^(th) Ed., revised in2011). All mice were obtained from either Charles River Laboratories orHarlan Laboratories.

Conjugate Preparation

The in vivo studies used a total of five siRNAs: four DMPK siRNAs thatwere shown to be cross-reactive with mouse in vitro (FIG. 17) and onesiRNA with a scrambled sequence that does not produce DMPK knockdown andcan be used as a negative control. All siRNAs were synthesized usingstandard solid phase synthesis methods that are described in theoligonucleotide synthesis literature. The single strands were purifiedby HPLC using standard methods and then pure single strands were mixedat equimolar ratios to generate pure duplexes. All siRNAs weresynthesized with a hexylamine linker on the 5′ end of the passenger(sense) strand that can act as a conjugation handle for linkage to theantibody. The siRNAs were synthesized using optimal base, sugar, andphosphate modifications that are well described in the field of RNAi tomaximize the potency, maximize the metabolic stability, and minimize theimmunogenicity of the duplex.

The anti-mouse transferrin receptor (TfR1, also known as CD71)monoclonal antibody (mAb) is a rat IgG2a subclass monoclonal antibodythat binds mouse CD71 protein with high affinity. This CD71 antibody wasproduced by BioXcell and it is commercially available (Catalog #BE0175).The antibody-siRNA conjugates were synthesized using the CD71 mAb fromBioXcell and the respective DMPK or scramble siRNAs. All conjugates weresynthesized through cysteine conjugation to the antibody and amineconjugation to the siRNA (through the hexylamine) utilizing abismaleimide-TFP ester linker as previously described. All conjugateswere purified by strong cation exchange (SAX) to isolate only theconjugate with a drug-antibody ratio (DAR) equal to 1 (i.e. a molarratio of 1 siRNA per mAb), as previously described. All antibody-siRNAconjugates were formulated by dilution in PBS for in vivo dosing.

In Vivo Dosing and Analysis

Purified DAR1 antibody-siRNA conjugates were dosed into groups (n=4) offemale wild-type CD-1 mice (4-6 weeks old) at 0.1, 0.3, 1, and 3 mg/kg(based on the weight of siRNA) by a single i.v. bolus injection into thetail vein at a dosing volume of 5 mL/kg. A single sham dose of PBSvehicle was injected at matched dose volumes into a control group (n=5)of female wild-type CD-1 mice (also 4-6 weeks old). The mice weresacrificed by CO₂ asphyxiation 7 days post-dose and 20-30 mg pieces ofmultiple tissues (gastrocnemius, tibialis anterior, quadriceps,diaphragm, heart, and liver) were harvested from each mouse andsnap-frozen in liquid nitrogen. TRIzol® reagent (Life Technologies) wasadded and then each tissue piece was homogenized using a TissueLyser II(Qiagen). RNA was isolated using the Direct-zol-96 RNA Kit (ZymoResearch) according to the manufacturer's instructions. 10 μl of RNA wasreverse transcribed to cDNA using the High Capacity cDNA ReverseTranscription Kit (Applied Biosystems) according to the manufacturer'sinstructions. cDNA samples were evaluated by qPCR with DMPK-specific andPPIB-specific TaqMan mouse gene expression probes (Thermo Fisher) usingTaqMan® Fast Advanced Master Mix (Applied Biosystems). DMPK values werenormalized within each sample to PPIB gene expression. Thequantification of DMPK downregulation was performed using the standard2^(−ΔΔC) method by comparing the treated animals to the PBS controlgroup. The in vivo DMPK mRNA knockdown results are presented in FIG.18A-FIG. 18F. All four DMPK siRNAs (the numbers indicated in the FIG.18A-FIG. 18F legends correspond to the ID # that is listed in Table 19(Tables 19A-19B)) were shown to effectively reduce levels of DMPK mRNAin all skeletal muscles that were analyzed (gastrocnemius, tibialisanterior, quadriceps, and diaphragm) in a dose-dependent manner. Themost active siRNA achieved greater than 75% DMPK mRNA knockdown in allskeletal muscles at the highest dose (3 mg/kg). The in vivo DMPKknockdown observed in skeletal muscles of mice (FIG. 18A-FIG. 18F)correlated well with the in vitro DMPK knockdown observed in the mouseC2C12 myoblast cell line (FIG. 17), with siRNA ID #s 2628 and 2636demonstrating higher mRNA knockdown than siRNA ID #s 535 and 1028. Inaddition to DMPK mRNA knockdown in skeletal muscle, strong activity(greater than 50% mRNA knockdown) was observed in mouse cardiac muscle(heart) as well. Finally, poor activity (less than 50% mRNA knockdown)was observed in mouse liver. These results demonstrate that it ispossible to achieve robust DMPK mRNA knockdown in multiple mouse musclegroups (including both skeletal and cardiac), while minimizing theknockdown in off-target tissues such as the liver.

Example 20: siRNA Synthesis

All siRNA single strands were fully assembled on solid phase usingstandard phospharamidite chemistry and purified using HPLC. Base, sugarand phosphate modifications that are well described in the field of RNAiwere used to optimize the potency of the duplex and reduceimmunogenicity. All the siRNA passenger strands contained a C6-NH₂conjugation handle on the 5′ end, see FIG. 20A-FIG. 21B. For the 21merduplex with 19 bases of complementarity and 3′ dinucleotide overhangs,the conjugation handle was connected to siRNA passenger strand via aninverted abasic phosphodiester, see FIG. 20A-FIG. 20B for thestructures. For the blunt ended duplex with 19 bases of complementarityand one 3′ dinucleotide overhang the conjugation handle was connected tosiRNA passenger strand via a phosphodiester on the terminal base, seeFIG. 21A-FIG. 21B for the structures.

Purified single strands were duplexed to get the double stranded siRNA.

Example 21: 2017-PK-401-C57BL6: In Vivo Transferrin mAb ConjugateDelivery of Various Atrogin-1 siRNAs

For groups 1-4, see study design in FIG. 22, the 21mer Atrogin-1 guidestrand was designed. The sequence (5′ to 3′) of the guide/antisensestrand was UCUACGUAGUUGAAUCUUCUU (SEQ ID NO: 14230). The guide and fullycomplementary RNA passenger strands were assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Base, sugarand phosphate modifications that are well described in the field of RNAiwere used to optimize the potency of the duplex and reduceimmunogenicity. Purified single strands were duplexed to get the doublestranded siRNA described in FIG. 20B. The passenger strand contained twoconjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end.Both conjugation handles were connected to siRNA passenger strand viaphosphodiester-inverted abasic-phosphodiester linker. Because the freethiol was not being used for conjugation, it was end capped withN-ethylmaleimide.

Antibody siRNA Conjugate Synthesis Using Bis-Maleimide (BisMal) Linker

Step 1: Antibody Reduction with TCEP

Antibody was buffer exchanged with 25 mM borate buffer (pH 8) with 1 mMDTPA and made up to 10 mg/ml concentration. To this solution, 4equivalents of TCEP in the same borate buffer were added and incubatedfor 2 hours at 37° C. The resultant reaction mixture was combined with asolution of BisMal-siRNA (1.25 equivalents) in pH 6.0 10 mM acetatebuffer at RT and kept at 4° C. overnight. Analysis of the reactionmixture by analytical SAX column chromatography showed antibody siRNAconjugate along with unreacted antibody and siRNA. The reaction mixturewas treated with 10 EQ of N-ethylmaleimide (in DMSO at 10 mg/mL) to capany remaining free cysteine residues.

Step 2: Purification

The crude reaction mixture was purified by AKTA Pure FPLC using anionexchange chromatography (SAX) method-1. Fractions containing DAR1 andDAR2 antibody-siRNA conjugates were isolated, concentrated, and bufferexchanged with pH 7.4 PBS.

Anion Exchange Chromatography Method (SAX)-1.

Column: Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5 mm ID×15 cm, 13 um

Solvent A: 20 mM TRIS buffer, pH 8.0; Solvent B: 20 mM TRIS, 1.5 M NaCl,pH 8.0; Flow Rate: 6.0 ml/min

Gradient:

a. % A % B Column Volume b. 1000 1 c. 81 19 0.5 d. 50 50 13 e. 40 60 0.5f. 0 100 0.5 g. 1000 2

Anion Exchange Chromatography (SAX) Method-2

Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm

Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80% 10 mM TRISpH 8, 20% ethanol, 1.5 M NaCl; Flow Rate: 0.75 ml/min

Gradient:

a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d. 11.00 40 60 e. 14.00 40 60f. 15.00 20 80 g. 16.00 90 10 h. 20.00 90 10

Step-3: Analysis of the Purified Conjugate

The purity of the conjugate was assessed by analytical HPLC using anionexchange chromatography method-2. For conjugate mTfR1-mAb-Atrogin-1(DAR1), the SAX retention time was 9.1 min and % purity (bychromatographic peak area) was 99.

In Vivo Study Design

The conjugates were assessed for their ability to mediate mRNAdownregulation of Atrogin-1 in skeletal muscle, in an in vivo experiment(C57BL6 mice). Mice were dosed via intravenous (iv) injection with PBSvehicle control and the indicated ASCs and doses, see FIG. 22. After theindicated time points, gastrocnemius (gastroc) and heart muscle tissueswere harvested and snap-frozen in liquid nitrogen. mRNA knockdown intarget tissue was determined using a comparative qPCR assay as describedin the methods section. Total RNA was extracted from the tissue, reversetranscribed and mRNA levels were quantified using TaqMan qPCR, using theappropriately designed primers and probes. PPIB (housekeeping gene) wasused as an internal RNA loading control, results were calculated by thecomparative Ct method, where the difference between the target gene Ctvalue and the PPIB Ct value (ΔCt) is calculated and then furthernormalized relative to the PBS control group by taking a seconddifference (ΔΔCt).

Results

The Atrogin-1 siRNA guide strands was able to mediate downregulation ofthe target gene in gastroc and heart muscle when conjugated to ananti-TfR mAb targeting the transferrin receptor, see FIG. 23 and FIG.24.

Conclusions

In this example, it was demonstrated that a TfR1-siAtrogin-1 conjugate,after in vivo delivery, mediated specific down regulation of the targetgene in gastroc and heart muscle. The ASC was made with ananti-transferrin antibody, mouse gastroc and heart muscle expresses thetransferrin receptor and the conjugate has a mouse specificanti-transferrin antibody to target the siRNA, resulting in accumulationof the conjugates in gastroc and heart muscle. Receptor mediate uptakeresulted in siRNA mediated knockdown of the target mRNA.

Example 22: 2017-PK-413-C57BL6: In Vivo Transferrin mAb ConjugateDelivery of Various MuRF1 Sequence

For groups 1-2, see study design in FIG. 25, the 21mer MuRF1 (2089)guide strand was designed. The sequence (5′ to 3′) of theguide/antisense strand was UUUCGCACCAACGUAGAAAUU (SEQ ID NO: 14231). Theguide and fully complementary RNA passenger strands were assembled onsolid phase using standard phospharamidite chemistry and purified overHPLC. Base, sugar and phosphate modifications that are well described inthe field of RNAi were used to optimize the potency of the duplex andreduce immunogenicity. Purified single strands were duplexed to get thedouble stranded siRNA described in FIG. 20B. The passenger strandcontained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH atthe 3′ end. Both conjugation handles were connected to siRNA passengerstrand via phosphodiester-inverted abasic-phosphodiester linkers.Because the free thiol was not being used for conjugation, it was endcapped with N-ethylmaleimide.

For groups 3-6, see study design in figure G, the 21mer MuRF1 (2265)guide strand was designed. The sequence (5′ to 3′) of theguide/antisense strand was UCGUGAGACAGUAGAUGUUUU (SEQ ID NO: 14232). Theguide and fully complementary RNA passenger strands were assembled onsolid phase using standard phospharamidite chemistry and purified overHPLC. Base, sugar and phosphate modifications that are well described inthe field of RNAi were used to optimize the potency of the duplex andreduce immunogenicity. Purified single strands were duplexed to get thedouble stranded siRNA described in FIG. 20B. The passenger strandcontained a single conjugation handle, a C6-NH₂ at the 5′ end connectedto siRNA passenger strand via phosphodiester-invertedabasic-phosphodiester linker.

For groups 7-10, see study design in figure G, the 21mer MuRF1 (2266)guide strand was designed. The sequence (5′ to 3′) of theguide/antisense strand was UCACACGUGAGACAGUAGAUU (SEQ ID NO: 14233). Theguide and fully complementary RNA passenger strands were assembled onsolid phase using standard phospharamidite chemistry and purified overHPLC. Base, sugar and phosphate modifications that are well described inthe field of RNAi were used to optimize the potency of the duplex andreduce immunogenicity. Purified single strands were duplexed to get thedouble stranded siRNA described in FIG. 20B. The passenger strandcontained a single conjugation handle, a C6-NH₂ at the 5′ end connectedto siRNA passenger strand via phosphodiester-invertedabasic-phosphodiester linker.

For groups 11-14, see study design in figure G, the 21mer MuRF1 (2267)guide strand was designed. The sequence (5′ to 3′) of theguide/antisense strand was UUCACACGUGAGACAGUAGUU (SEQ ID NO: 14234). Theguide and fully complementary RNA passenger strands were assembled onsolid phase using standard phospharamidite chemistry and purified overHPLC. Base, sugar and phosphate modifications that are well described inthe field of RNAi were used to optimize the potency of the duplex andreduce immunogenicity. Purified single strands were duplexed to get thedouble stranded siRNA described in FIG. 20B. The passenger strandcontained a single conjugation handle, a C6-NH₂ at the 5′ end connectedto siRNA passenger strand via phosphodiester-invertedabasic-phosphodiester linker.

For groups 15-18, see study design in figure G, the 21mer MuRF1 (2268)guide strand was designed. The sequence (5′ to 3′) of theguide/antisense strand was UAAUAUUUCAUUUCGCACCUU (SEQ ID NO: 14235). Theguide and fully complementary RNA passenger strands were assembled onsolid phase using standard phospharamidite chemistry and purified overHPLC. Base, sugar and phosphate modifications that are well described inthe field of RNAi were used to optimize the potency of the duplex andreduce immunogenicity. Purified single strands were duplexed to get thedouble stranded siRNA described in FIG. 20B. The passenger strandcontained a single conjugation handle, a C6-NH₂ at the 5′ end connectedto siRNA passenger strand via phosphodiester-invertedabasic-phosphodiester linker.

For groups 19-22, see study design in figure G, the 21mer MuRF1 (2269)guide strand was designed. The sequence (5′ to 3′) of theguide/antisense strand was UAAGCACCAAAUUGGCAUAUU (SEQ ID NO: 14236). Theguide and fully complementary RNA passenger strands were assembled onsolid phase using standard phospharamidite chemistry and purified overHPLC. Base, sugar and phosphate modifications that are well described inthe field of RNAi were used to optimize the potency of the duplex andreduce immunogenicity. Purified single strands were duplexed to get thedouble stranded siRNA described in FIG. 20B. The passenger strandcontained a single conjugation handle, a C6-NH₂ at the 5′ end connectedto siRNA passenger strand via phosphodiester-invertedabasic-phosphodiester linker.

Antibody siRNA Conjugate Synthesis Using Bis-Maleimide (BisMal) Linker

Step 1: Antibody Reduction with TCEP

Antibody was buffer exchanged with 25 mM borate buffer (pH 8) with 1 mMDTPA and made up to 10 mg/ml concentration. To this solution, 4equivalents of TCEP in the same borate buffer were added and incubatedfor 2 hours at 37° C. The resultant reaction mixture was combined with asolution of BisMal-siRNA (1.25 equivalents) in pH 6.0 10 mM acetatebuffer at RT and kept at 4° C. overnight. Analysis of the reactionmixture by analytical SAX column chromatography showed antibody siRNAconjugate along with unreacted antibody and siRNA. The reaction mixturewas treated with 10 EQ of N-ethylmaleimide (in DMSO at 10 mg/mL) to capany remaining free cysteine residues.

Step 2: Purification

The crude reaction mixture was purified by AKTA Pure FPLC using anionexchange chromatography (SAX) method-1. Fractions containing DAR1 andDAR2 antibody-siRNA conjugates were isolated, concentrated and bufferexchanged with pH 7.4 PBS.

Anion Exchange Chromatography Method (SAX)-1.

Column: Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5 mm ID×15 cm, 13 um

Solvent A: 20 mM TRIS buffer, pH 8.0; Solvent B: 20 mM TRIS, 1.5 M NaCl,pH 8.0; Flow Rate: 6.0 ml/min

Gradient:

a. % A % B Column Volume b. 100 0 1 c. 81 19 0.5 d. 50 50 13 e. 40 600.5 f. 0 100 0.5 g. 100 0 2

Anion Exchange Chromatography (SAX) Method-2

Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm

Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80% 10 mM TRISpH 8, 20% ethanol, 1.5 M NaCl; Flow Rate: 0.75 ml/min

Gradient:

a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d. 11.00 40 60 e. 14.00 40 60f. 15.00 20 80 g. 16.00 90 10 h. 20.00 90 10

Step-3: Analysis of the Purified Conjugate

The purity of the conjugate was assessed by analytical HPLC using anionexchange chromatography method-2 (Table 19).

TABLE 19 SAX retention % purity (by Conjugate time (mm) peak area)mTfR1-mAb-MuRF1(R2089) (DAR1) 9.3 99 mTfR1-mAb-MuRF1(R2265) (DAR1) 9.195 mTfR1-mAb-MuRF1(R2266) (DAR1) 9.1 98 mTfR1-mAb-MuRF1(R2267) (DAR1)9.1 98 mTfR1-mAb-MuRF1(R2268) (DAR1) 9.1 97 mTfR1-mAb-MuRF1(R2269)(DAR1) 9.2 97

In Vivo Study Design

The conjugates were assessed for their ability to mediate mRNAdownregulation of MuRF1 in muscle (gastroc and heart), in an in vivoexperiment (C57BL6 mice). Mice were dosed via intravenous (iv) injectionwith PBS vehicle control and the indicated ASCs and doses, see FIG. 25.After 96 hours, gastrocnemius (gastroc) and heart muscle tissues wereharvested and snap-frozen in liquid nitrogen. mRNA knockdown in targettissue was determined using a comparative qPCR assay as described in themethods section. Total RNA was extracted from the tissue, reversetranscribed and mRNA levels were quantified using TaqMan qPCR, using theappropriately designed primers and probes. PPIB (housekeeping gene) wasused as an internal RNA loading control, results were calculated by thecomparative Ct method, where the difference between the target gene Ctvalue and the PPIB Ct value (ΔCt) is calculated and then furthernormalized relative to the PBS control group by taking a seconddifference (ΔΔCt).

Results

The MuRF1 siRNA guide strands was able to mediate downregulation of thetarget gene in gastroc and heart muscle when conjugated to an anti-TfR1mAb targeting the transferrin receptor 1, see FIG. 26 and FIG. 27.

Conclusions

In this example, it was demonstrated that TfR1-MuRF1 conjugates, afterin vivo delivery, mediated specific down regulation of the target genein gastroc and heart muscle. The ASC was made with an anti-transferrin1antibody, mouse gastroc and heart muscle expresses the transferrinreceptor1 and the conjugate has a mouse specific anti-transferrinantibody to target the siRNA, resulting in accumulation of theconjugates in gastroc muscle. Receptor mediate uptake resulted in siRNAmediated knockdown of the target mRNA.

Example 23: 2017-PK-412-C57BL6: Prevention of Dexamethasone InduceMuscle Atrophy with Atrogin-1 and MuRF1 TfR1-mAb Conjugates

For this experiment three different siRNAs were used:

(1): A 21mer Atrogin-1 guide strand was designed. The sequence (5′ to3′) of the guide/antisense strand was UCUACGUAGUUGAAUCUUCUU (SEQ ID NO:14230). The guide and fully complementary RNA passenger strands wereassembled on solid phase using standard phospharamidite chemistry andpurified over HPLC. Base, sugar and phosphate modifications that arewell described in the field of RNAi were used to optimize the potency ofthe duplex and reduce immunogenicity. Purified single strands wereduplexed to get the double stranded siRNA described in FIG. 20B. Thepassenger strand contained two conjugation handles, a C6-NH₂ at the 5′end and a C6-SH at the 3′ end. Both conjugation handles were connectedto siRNA passenger strand via phosphodiester-invertedabasic-phosphodiester linkers. Because the free thiol was not being usedfor conjugation, it was end capped with N-ethylmaleimide.

(2): A 21mer MuRF1 guide strand was designed. The sequence (5′ to 3′) ofthe guide/antisense strand was UUUCGCACCAACGUAGAAAUU (SEQ ID NO: 14231).The guide and fully complementary RNA passenger strands were assembledon solid phase using standard phospharamidite chemistry and purifiedover HPLC. Base, sugar and phosphate modifications that are welldescribed in the field of RNAi were used to optimize the potency of theduplex and reduce immunogenicity. Purified single strands were duplexedto get the double stranded siRNA described in FIG. 20B. The passengerstrand contained two conjugation handles, a C6-NH₂ at the 5′ end and aC6-SH at the 3′ end. Both conjugation handles were connected to siRNApassenger strand via phosphodiester-inverted abasic-phosphodiesterlinkers. Because the free thiol was not being used for conjugation, itwas end capped with N-ethylmaleimide.

(3): Negative control siRNA sequence (scramble): A published (Burke etal. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 14228). The same base, sugar and phosphate modifications that wereused for the active MSTN siRNA duplex were used in the negative controlsiRNA. All siRNA single strands were fully assembled on solid phaseusing standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphodiester-invertedabasic-phosphodiester linker. Because the free thiol was not being usedfor conjugation, it was end capped with N-ethylmaleimide.

Antibody siRNA Conjugate Synthesis Using Bis-Maleimide (BisMal) Linker

Step 1: Antibody Reduction with TCEP

Antibody was buffer exchanged with 25 mM borate buffer (pH 8) with 1 mMDTPA and made up to 10 mg/ml concentration. To this solution, 4equivalents of TCEP in the same borate buffer were added and incubatedfor 2 hours at 37° C. The resultant reaction mixture was combined with asolution of BisMal-siRNA (1.25 equivalents) in pH 6.0 10 mM acetatebuffer at RT and kept at 4° C. overnight. Analysis of the reactionmixture by analytical SAX column chromatography showed antibody siRNAconjugate along with unreacted antibody and siRNA. The reaction mixturewas treated with 10 EQ of N-ethylmaleimide (in DMSO at 10 mg/mL) to capany remaining free cysteine residues.

Step 2: Purification

The crude reaction mixture was purified by AKTA Pure FPLC using anionexchange chromatography (SAX) method-1. Fractions containing DAR1 andDAR2 antibody-siRNA conjugates were isolated, concentrated and bufferexchanged with pH 7.4 PBS.

Anion Exchange Chromatography Method (SAX)-1.

Column: Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5 mm ID×15 cm, 13 um

Solvent A: 20 mM TRIS buffer, pH 8.0; Solvent B: 20 mM TRIS, 1.5 M NaCl,pH 8.0; Flow Rate: 6.0 ml/min

Gradient:

a. % A % B Column Volume b. 100 0 1 c. 81 19 0.5 d. 50 50 13 e. 40 600.5 f. 0 100 0.5 g. 100 0 2

Anion Exchange Chromatography (SAX) Method-2

Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm

Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80% 10 mM TRISpH 8, 20% ethanol, 1.5 M NaCl; Flow Rate: 0.75 ml/min

Gradient:

a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d. 11.00 40 60 e. 14.00 40 60f. 15.00 20 80 g. 16.00 90 10 h. 20.00 90 10

Step-3: Analysis of the Purified Conjugate

The purity of the conjugate was assessed by analytical HPLC using anionexchange chromatography method-2 (Table 20).

TABLE 20 SAX retention % purity (by Conjugate time (min) peak area)mTfR1-Atrogin-1 (DAR1) 9.3 97 mTfR1-MuRF1 (DAR1) 9.5 98 mTfR1-SC (DAR1)9.0 99

In Vivo Study Design

The conjugates were assessed for their ability to mediate mRNAdownregulation of MuRF1 and Atrogin-1 in muscle (gastroc) in thepresence and absence of muscle atrophy, in an in vivo experiment (C57BL6mice). Mice were dosed via intravenous (iv) injection with PBS vehiclecontrol and the indicated ASCs and doses, see Table 21. Seven days postconjugate delivery, for groups 2-4, 9-11, and 16-18, muscle atrophy wasinduced by the daily administration, via intraperitoneal injection (10mg/kg) of dexamethasone for 21 days. For the control groups 5-7, 12-14and 19-21 (no induction of muscle atrophy) PBS was administered by thedaily intraperitoneal injection. Groups 1, 8, 15 and 22 were harvestedat day 7 to establish the baseline measurements of mRNA expression andmuscle weighted, prior to induction of muscle atrophy. At the timepoints indicated, gastrocnemius (gastroc) and heart muscle tissues wereharvested, weighed and snap-frozen in liquid nitrogen. mRNA knockdown intarget tissue was determined using a comparative qPCR assay as describedin the methods section. Total RNA was extracted from the tissue, reversetranscribed and mRNA levels were quantified using TaqMan qPCR, using theappropriately designed primers and probes. PPIB (housekeeping gene) wasused as an internal RNA loading control, results were calculated by thecomparative Ct method, where the difference between the target gene Ctvalue and the PPIB Ct value (ΔCt) is calculated and then furthernormalized relative to the PBS control group by taking a seconddifference (ΔΔCt).

Quantitation of tissue siRNA concentrations was determined using astem-loop qPCR assay as described in the methods section. The antisensestand of the siRNA was reverse transcribed using a TaGMan MicroRNAreverse transcription kit using a sequence-specific stem-loop RT primer.The cDNA from the RT step was then utilized for real-time PCR and Ctvalues were transformed into plasma or tissue concentrations using thelinear equations derived from the standard curves.

TABLE 21 Dex/PBS Dosing Compound Info Dose siRNA Harvest Animal andGroup Info Volume # of Dose Dose # of Time Group Test Article N ROA(mL/kg) Doses Schedule (mg/kg) Doses (d) 1 mTfR1-Atrogin-1 5 — — — — 3 17 (DAR1) 2 mTfR1-Atrogin-1 5 IP 6.25 Daily; 192 h 3 1 10 (DAR1), +DEX 21Post (10 mg/kg) Days ASC 3 mTfR1-Atrogin-1 5 IP 6.25 Daily; 192 h 3 1 17(DAR1), +DEX 21 Post (10 mg/kg) Days ASC 4 mTfR1-Atrogin-1 5 IP 6.25Daily; 192 h 3 1 28 (DAR1), +DEX 21 Post (10 mg/kg) Days ASC 5mTfR1-Atrogin-1 5 IP 6.25 Daily; 192 h 3 1 10 (DAR1), PBS 21 Post DaysASC 6 mTfR1-Atrogin-1 5 IP 6.25 Daily; 192 h 3 1 17 (DAR1), PBS 21 PostDays ASC 7 mTfR1-Atrogin-1 5 IP 6.25 Daily; 192 h 3 1 28 (DAR1), PBS 21Post Days ASC 8 mTfR1-Atrogin-1 5 — — — — 3 + 3 1 7 (DAR1) + mTfR1-MuRF1(DAR1) 9 mTfR1-Atrogin-1 5 IP 6.25 Daily; 192 h 3 + 3 1 10 (DAR1) + 21Post mTfR1-MuRF1 Days ASC (DAR1), +DEX (10 mg/kg) 10 mTfR1-Atrogin-1 5IP 6.25 Daily; 192 h 3 + 3 1 17 (DAR1) + 21 Post mTfR1-MuRF1 Days ASC(DAR1), +DEX (10 mg/kg) 11 mTfR1-Atrogin-1 5 IP 6.25 Daily; 192 h 3 + 31 28 (DAR1) + 21 Post mTfR1-MuRF1 Days ASC (DAR1), +DEX (10 mg/kg) 12mTfR1-Atrogin-1 5 IP 6.25 Daily; 192 h 3 + 3 1 10 (DAR1) + 21 PostmTfR1-MuRF1 Days ASC (DAR1), +PBS 13 mTfR1-Atrogin-1 5 IP 6.25 Daily;192 h 3 + 3 1 17 (DAR1) + 21 Post mTfR1-MuRF1 Days ASC (DAR1), +PBS 14mTfR1-Atrogin-1 5 IP 6.25 Daily; 192 h 3 + 3 1 28 (DAR1) + 21 PostmTfR1-MuRF1 Days ASC (DAR1), +PBS 15 mTfR1-SC 5 — — — — 3 1 7 (DAR1) 16mTfR1-SC 5 IP 6.25 Daily; 192 h 3 1 10 DAR1), +DEX 21 Post (10 mg/kg)Days ASC 17 mTfR1-SC 5 IP 6.25 Daily; 192 h 3 1 17 (DAR1), +DEX 21 Post(10 mg/kg) Days ASC 18 mTfR1-SC 5 IP 6.25 Daily; 192 h 3 1 28 (DAR1),+DEX 21 Post (10 mg/kg) Days ASC 19 mTfR1-SC 5 IP 6.25 Daily; 192 h 3 110 (DAR1), PBS 21 Post Days ASC 20 mTfR1-SC 5 IP 6.25 Daily; 192 h 3 117 (DAR1), PBS 21 Post Days ASC 21 mTfR1-SC 5 IP 6.25 Daily; 192 h 3 128 (DAR1), PBS 21 Post Days ASC 22 PBS Control 5 — — — — — 1 7

Results

The data are summarized in FIG. 28-FIG. 31. Co-delivery of Atrogin-1 andMuRF1 siRNAs efficiently downregulated Atrogin-1 and MuRF1 mRNAexpression in normal and atrophic muscles, when delivered using a TfR1mAb conjugate. Induction of atrophy transiently induces Atrogin-1 andMuRF1 expression about 4-fold. A single dose ofmTfR1-Atrogin-1+TfR1.mAb-siMuRF1 (3 mg/kg, each and dose as a mixture)reduced Atrogin-1 and MuRF1 mRNA levels by >70% in normal and atrophicgastrocnemius muscle. Downregulation of MuRF1 and Atrogin-1 mRNAincreases gastrocnemius weight by 5-10% and reduces DEX-inducedgastrocnemius weight loss by 50%. Downregulation of Atrogin-1 alone hasno significant effect on gastrocnemius weight changes. In the absence ofmuscle atrophy treatment with Atrogin-1/MuRF1 siRNAs induces musclehypertrophy.

Conclusions

In this example, it was demonstrated that co-delivery of Atrogin-1 andMuRF1 siRNAs efficiently downregulated Atrogin-1 and MuRF1 mRNAexpression in normal and atrophic gastroc muscles, when delivered usinga TfR1 mAb conjugate. The conjugates had little effect on heart muscle,where downregulation of Atrogin-1 could be detrimental. Downregulationof MuRF1 and Atrogin-1 mRNA increased gastroc muscle weight by 5-10% andreduced DEX-induced gastroc muscle weight loss by 50%. Downregulation ofAtrogin-1 alone has no significant effect on gastrocnemius weightchanges. The ASC were made with an anti-transferrin antibody, mousegastroc muscle expresses the transferrin receptor and the conjugate hasa mouse specific anti-transferrin antibody to target the siRNA,resulting in accumulation of the conjugates in gastroc muscle. Receptormediate uptake resulted in siRNA mediated knockdown of the target mRNA.

Example 24: 2017-PK-435-C57BL6: In Vivo Dose Response Experiment forTransferrin mAb Conjugate Delivery of Atrogin-1

For groups 1-12, see study design in FIG. 32, the 21mer Atrogin-1 guidestrand was designed. The sequence (5′ to 3′) of the guide/antisensestrand was UCGUAGUUAAAUCUUCUGGUU (SEQ ID NO: 14237). The guide and fullycomplementary RNA passenger strands were assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Base, sugarand phosphate modifications that are well described in the field of RNAiwere used to optimize the potency of the duplex and reduceimmunogenicity. Purified single strands were duplexed to get the doublestranded siRNA described in figure A. The passenger strand contained twoconjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end.Both conjugation handles were connected to siRNA passenger strand viaphosphodiester-inverted abasic-phosphodiester linkers. Because the freethiol was not being used for conjugation, it was end capped withN-ethylmaleimide.

For groups 13-18 see study design in FIG. 32, a 21mer negative controlsiRNA sequence (scramble) (published by Burke et al. (2014) Pharm. Res.,31(12):3445-60) with 19 bases of complementarity and 3′ dinucleotideoverhangs was used. The sequence (5′ to 3′) of the guide/antisensestrand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 14228). The same base,sugar and phosphate modifications that were used for the active MSTNsiRNA duplex were used in the negative control siRNA. All siRNA singlestrands were fully assembled on solid phase using standardphospharamidite chemistry and purified over HPLC. Purified singlestrands were duplexed to get the double stranded siRNA. The passengerstrand contained two conjugation handles, a C6-NH₂ at the 5′ end and aC6-SH at the 3′ end. Both conjugation handles were connected to siRNApassenger strand via phosphodiester-inverted abasic-phosphodiesterlinker. Because the free thiol was not being used for conjugation, itwas end capped with N-ethylmaleimide.

Antibody siRNA Conjugate Synthesis Using Bis-Maleimide (BisMal) Linker

Step 1: Antibody Reduction with TCEP

Antibody was buffer exchanged with 25 mM borate buffer (pH 8) with 1 mMDTPA and made up to 10 mg/ml concentration. To this solution, 4equivalents of TCEP in the same borate buffer were added and incubatedfor 2 hours at 37° C. The resultant reaction mixture was combined with asolution of BisMal-siRNA (1.25 equivalents) in pH 6.0 10 mM acetatebuffer at RT and kept at 4° C. overnight. Analysis of the reactionmixture by analytical SAX column chromatography showed antibody siRNAconjugate along with unreacted antibody and siRNA. The reaction mixturewas treated with 10 EQ of N-ethylmaleimide (in DMSO at 10 mg/mL) to capany remaining free cysteine residues.

Step 2: Purification

The crude reaction mixture was purified by AKTA Pure FPLC using anionexchange chromatography (SAX) method-1. Fractions containing DAR1 andDAR2 antibody-siRNA conjugates were isolated, concentrated and bufferexchanged with pH 7.4 PBS.

Anion Exchange Chromatography Method (SAX)-1.

Column: Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5 mm ID×15 cm, 13 um

Solvent A: 20 mM TRIS buffer, pH 8.0; Solvent B: 20 mM TRIS, 1.5 M NaCl,pH 8.0; Flow Rate: 6.0 ml/min

Gradient:

a. % A % B Column Volume b. 100 0 1 c. 81 19 0.5 d. 50 50 13 e. 40 600.5 f. 0 100 0.5 g. 100 0 2

Anion Exchange Chromatography (SAX) Method-2

Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm

Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80% 10 mM TRISpH 8, 20% ethanol, 1.5 M NaCl; Flow Rate: 0.75 ml/min

Gradient:

a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d. 11.00 40 60 e. 14.00 40 60f. 15.00 20 80 g. 16.00 90 10 h. 20.00 90 10

Step-3: Analysis of the Purified Conjugate

The purity of the conjugate was assessed by analytical HPLC using anionexchange chromatography method-2 (Table 22).

TABLE 22 SAX retention % purity (by Conjugate time (min) peak area)TfR1-Atrogin-1 DAR1 9.2 99 TfR1-Scramble DAR1 8.9 93

In Vivo Study Design

The conjugates were assessed for their ability to mediate mRNAdownregulation of Atrogin-1 in muscle (gastroc) in the presence andabsence of muscle atrophy, in an in vivo experiment (C57BL6 mice). Micewere dosed via intravenous (iv) injection with PBS vehicle control andthe indicated ASCs and doses, see FIG. 32. Seven days post conjugatedelivery, for groups 3, 6, 9, 12, and 15, muscle atrophy was induced bythe daily administration via intraperitoneal injection (10 mg/kg) ofdexamethasone for 3 days. For the control groups 2, 5, 8, 11, and 14 (noinduction of muscle atrophy) PBS was administered by the dailyintraperitoneal injection. Groups 1, 4, 7, 10, and 13 were harvested atday 7 to establish the baseline measurements of mRNA expression andmuscle weighted, prior to induction of muscle atrophy. At three dayspost-atrophy induction (or 10 days post conjugate delivery),gastrocnemius (gastroc) muscle tissues were harvested, weighed andsnap-frozen in liquid nitrogen. mRNA knockdown in target tissue wasdetermined using a comparative qPCR assay as described in the methodssection. Total RNA was extracted from the tissue, reverse transcribedand mRNA levels were quantified using TaqMan qPCR, using theappropriately designed primers and probes. PPIB (housekeeping gene) wasused as an internal RNA loading control, results were calculated by thecomparative Ct method, where the difference between the target gene Ctvalue and the PPIB Ct value (ΔCt) is calculated and then furthernormalized relative to the PBS control group by taking a seconddifference (ΔΔCt).

Quantitation of tissue siRNA concentrations was determined using astem-loop qPCR assay as described in the methods section. The antisensestrand of the siRNA was reverse transcribed using a TaqMan MicroRNAreverse transcription kit using a sequence-specific stem-loop RT primer.The cDNA from the RT step was then utilized for real-time PCR and Ctvalues were transformed into plasma or tissue concentrations using thelinear equations derived from the standard curves.

Results

The data are summarized in FIG. 33-FIG. 35. The Atrogin-1 siRNA guidestrands were able to mediate downregulation of the target gene ingastroc muscle when conjugated to an anti-TfR mAb targeting thetransferrin receptor, see FIG. 33. Increasing the dose from 3 to 9 mg/kgreduced atrophy-induced Atrogin-1 mRNA levels 2-3 fold. The maximal KDachievable with this siRNA was 80% and a tissue concentration of 40 nMwas needed to achieve maximal KD in atrophic muscles. This highlightsthe conjugate delivery approach is able to change disease induce mRNAexpression levels of Atrogin-1 (see FIG. 34), by increasing theincreasing the dose. FIG. 35 highlights that mRNA down regulation ismediated by RISC loading of the Atrogin-1 guide strands and isconcentration dependent.

Conclusions

In this example, it was demonstrated that a TfR1-Atrogin-1 conjugates,after in vivo delivery, mediated specific down regulation of the targetgene in gastroc muscle in a dose dependent manner. After induction ofatrophy the conjugate was able to mediate disease induce mRNA expressionlevels of Atrogin-1 at the higher doses. Higher RISC loading of theAtrogin-1 guide strand correlated with increased mRNA downregulation.

Example 25: 2017-PK-381-C57BL6: Myostatin (MSTN) Downregulation ReducesMuscle Loss in Dexamethasone-Treated Mice

For groups 1-12, see study design in Table 24, the 21mer Atrogin-1 guidestrand was designed. The sequence (5′ to 3′) of the guide/antisensestrand was UCGUAGUUAAAUCUUCUGGUU (SEQ ID NO: 14237). The guide and fullycomplementary RNA passenger strands were assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Base, sugarand phosphate modifications that are well described in the field of RNAiwere used to optimize the potency of the duplex and reduceimmunogenicity. Purified single strands were duplexed to get the doublestranded siRNA described in FIG. 20B. The passenger strand contained twoconjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end.Both conjugation handles were connected to siRNA passenger strand viaphosphodiester-inverted abasic-phosphodiester linkers. Because the freethiol was not being used for conjugation, it was end capped withN-ethylmaleimide.

For groups 13-18 see study design in Table 24, a 21mer negative controlsiRNA sequence (scramble) (published by Burke et al. (2014) Pharm. Res.,31(12):3445-60) with 19 bases of complementarity and 3′ dinucleotideoverhangs were used. The sequence (5′ to 3′) of the guide/antisensestrand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 14228). The same base,sugar and phosphate modifications that were used for the active MSTNsiRNA duplex were used in the negative control siRNA. All siRNA singlestrands were fully assembled on solid phase using standardphospharamidite chemistry and purified over HPLC. Purified singlestrands were duplexed to get the double stranded siRNA. The passengerstrand contained two conjugation handles, a C6-NH₂ at the 5′ end and aC6-SH at the 3′ end. Both conjugation handles were connected to siRNApassenger strand via phosphodiester-inverted abasic-phosphodiesterlinker. Because the free thiol was not being used for conjugation, itwas end capped with N-ethylmaleimide.

Antibody siRNA Conjugate Synthesis Using Bis-Maleimide (BisMal) Linker

Step 1: Antibody Reduction with TCEP

Antibody was buffer exchanged with 25 mM borate buffer (pH 8) with 1 mMDTPA and made up to 10 mg/ml concentration. To this solution, 4equivalents of TCEP in the same borate buffer were added and incubatedfor 2 hours at 37° C. The resultant reaction mixture was combined with asolution of BisMal-siRNA (1.25 equivalents) in pH 6.0 10 mM acetatebuffer at RT and kept at 4° C. overnight. Analysis of the reactionmixture by analytical SAX column chromatography showed antibody siRNAconjugate along with unreacted antibody and siRNA. The reaction mixturewas treated with 10 EQ of N-ethylmaleimide (in DMSO at 10 mg/mL) to capany remaining free cysteine residues.

Step 2: Purification

The crude reaction mixture was purified by AKTA Pure FPLC using anionexchange chromatography (SAX) method-1. Fractions containing DAR1 andDAR2 antibody-siRNA conjugates were isolated, concentrated and bufferexchanged with pH 7.4 PBS.

Anion Exchange Chromatography Method (SAX)-1.

Column: Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5 mm ID×15 cm, 13 um

Solvent A: 20 mM TRIS buffer, pH 8.0; Solvent B: 20 mM TRIS, 1.5 M NaCl,pH 8.0; Flow Rate: 6.0 ml/min

Gradient:

a. % A % B Column Volume b. 100 0 1 c. 81 19 0.5 d. 50 50 13 e. 40 600.5 f. 0 100 0.5 g. 100 0 2

Anion Exchange Chromatography (SAX) Method-2

Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm

Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80% 10 mM TRISpH 8, 20% ethanol, 1.5 M NaCl; Flow Rate: 0.75 ml/min

Gradient:

a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d. 11.00 40 60 e. 14.00 40 60f. 15.00 20 80 g. 16.00 90 10 h. 20.00 90 10

Step-3: Analysis of the Purified Conjugate

The purity of the conjugate was assessed by analytical HPLC using anionexchange chromatography method-2 (Table 23).

TABLE 23 SAX retention % purity (by Conjugate time (min) peak area)mTfR1-MSTN (DAR1) 9.2 98 mTfR1-SC (DAR1) 8.9 98

In Vivo Study Design

The conjugates were assessed for their ability to mediate mRNAdownregulation of MSTN in muscle (gastroc) in the presence and absenceof muscle atrophy, in an in vivo experiment (C57BL6 mice). Mice weredosed via intravenous (iv) injection with PBS vehicle control and theindicated ASCs and doses, see Table 24. Seven days post conjugatedelivery, for groups 2, 3, 4, 9, 10 and 11, muscle atrophy was inducedby the daily administration via intraperitoneal injection (10 mg/kg) ofdexamethasone for 13 days. For the control groups 5, 6, 7, 12, 13 and 14(no induction of muscle atrophy), PBS was administered by the dailyintraperitoneal injection. Groups 1 and 8 were harvested at day 7 toestablish the baseline measurements of mRNA expression and muscleweighted, prior to induction of muscle atrophy. At 3, 7, and 14 dayspost-atrophy induction (or 10, 14, and 21 days post conjugate delivery),gastrocnemius (gastroc) muscle tissues were harvested, weighed andsnap-frozen in liquid nitrogen. mRNA knockdown in target tissue wasdetermined using a comparative qPCR assay as described in the methodssection. Total RNA was extracted from the tissue, reverse transcribedand mRNA levels were quantified using TaqMan qPCR, using theappropriately designed primers and probes. PPIB (housekeeping gene) wasused as an internal RNA loading control, results were calculated by thecomparative Ct method, where the difference between the target gene Ctvalue and the PPIB Ct value (ΔCt) is calculated and then furthernormalized relative to the PBS control group by taking a seconddifference (ΔΔCt).

Quantitation of tissue siRNA concentrations was determined using astem-loop qPCR assay as described in the methods section. The antisensestrand of the siRNA was reverse transcribed using a TaqMan MicroRNAreverse transcription kit using a sequence-specific stem-loop RT primer.The cDNA from the RT step was then utilized for real-time PCR and Ctvalues were transformed into plasma or tissue concentrations using thelinear equations derived from the standard curves.

TABLE 24 Animal Dex/PBS Dosing Compound Info and Group Info Dose siRNAHarvest Test Volume # of Dose Dose Time Group Article N ROA (mL/kg)Doses Schedule (mg/kg) ROA (d) 1 mTfR1- 5 — — — — 3 IV 7 MSTN (DAR1) 2mTfR1- 5 IP 6.25 Daily; 192 h Post 3 IV 10 MSTN 13 ASC (DAR1), Days +DEX(10 mg/kg) 3 mTfR1- 5 IP 6.25 Daily; 192 h Post 3 IV 14 MSTN 13 ASC (DAR= 1), Days +DEX (10 mg/kg) 4 mTfR1- 5 IP 6.25 Daily; 192 h Post 3 IV 21MSTN 13 ASC (DAR = 1), Days +DEX (10 mg/kg) 5 mTfR1- 5 IP 6.25 Daily;192 h Post 3 IV 10 MSTN 13 ASC (DAR1), Days PBS 6 mTfR1- 5 IP 6.25Daily; 192 h Post 3 IV 14 MSTN 13 ASC (DAR1), Days PBS 7 mTfR1- 5 IP6.25 Daily; 192 h Post 3 IV 21 MSTN 13 ASC (DAR1), Days PBS 8 mTfR1-SC 5— — — — 3 IV 7 (DAR1) 9 mTfR1-SC 5 IP 6.25 Daily; 192 h Post 3 IV 10(DAR1), 13 ASC +DEX Days (10 mg/kg) 10 mTfR1-SC 5 IP 6.25 Daily; 192 hPost 3 IV 14 (DAR1), 13 ASC +DEX Days (10 mg/kg) 11 mTfR1-SC 5 IP 6.25Daily; 192 h Post 3 IV 21 (DAR1), 13 ASC +DEX Days (10 mg/kg) 12mTfR1-SC 5 IP 6.25 Daily; 192 h Post 3 IV 10 (DAR1), 13 ASC PBS Days 13mTfR1-SC 5 IP 6.25 Daily; 192 h Post 3 IV 14 (DAR1), 13 ASC PBS Days 14mTfR1-SC 5 IP 6.25 Daily; 192 h Post 3 IV 21 (DAR1), 13 ASC PBS Days 15PBS 5 — — — — — IV 7 Control

Results

The data are summarized in FIG. 36 and FIG. 37. The MSTN siRNA guidestrands were able to mediate downregulation of the target gene ingastroc muscle when conjugated to an anti-TfR mAb targeting thetransferrin receptor, see FIG. 36, in the presence and absence ofdexamethasone induced atrophy. A single of 3 mg/kg siRNA downregulatedMSTN mRNA levels by >75%. In the presence of dexamethasone inducedatrophy, MSTN downregulation increased muscle mass and attenuatesDex-induced muscle loss, see FIG. 37.

Conclusions

In this example, it was demonstrated that a TfR1-MSTN conjugate, afterin vivo delivery, mediated specific down regulation of the target genein gastroc muscle. After induction of atrophy the conjugate was able toincrease muscle mass and attenuate Dex-induced muscle loss.

Example 26: 2017-PK-496-C57BL6: Atrogin-1 and MuRF1 DownregulationReduces Leg Muscle Loss Upon Sciatic Nerve Denervation in Mice

For groups 1-4, see study design in FIG. 38, the 21mer Atrogin-1 guidestrand was designed. The sequence (5′ to 3′) of the guide/antisensestrand was UUGGGUAACAUCGUACAAGUU (SEQ ID NO: 14238). The guide and fullycomplementary RNA passenger strands were assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Base, sugarand phosphate modifications that are well described in the field of RNAiwere used to optimize the potency of the duplex and reduceimmunogenicity. Purified single strands were duplexed to get the doublestranded siRNA described in FIG. 20B. The passenger strand contained twoconjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end.Both conjugation handles were connected to siRNA passenger strand viaphosphodiester-inverted abasic-phosphodiester linkers. Because the freethiol was not being used for conjugation, it was end capped withN-ethylmaleimide.

For groups 5-6, see study design in figure V, the 21mer MuRF1 guidestrand was designed. The sequence (5′ to 3′) of the guide/antisensestrand was UUUCGCACCAACGUAGAAAUU (SEQ ID NO: 14231). The guide and fullycomplementary RNA passenger strands were assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Base, sugarand phosphate modifications that are well described in the field of RNAiwere used to optimize the potency of the duplex and reduceimmunogenicity. Purified single strands were duplexed to get the doublestranded siRNA described in FIG. 20B. The passenger strand contained twoconjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end.Both conjugation handles were connected to siRNA passenger strand viaphosphodiester-inverted abasic-phosphodiester linkers. Because the freethiol was not being used for conjugation, it was end capped withN-ethylmaleimide.

For groups 7-12, the Atrogin-1 and MuRF1 were design as above. Afterconjugation to the TfR1 mAb and after purification and isolation of theindividual DAR1 species, were mixed and co-administered.

For group 13, see study design in FIG. 38, a 21mer negative controlsiRNA sequence (scramble) (published by Burke et al. (2014) Pharm. Res.,31(12):3445-60) with 19 bases of complementarity and 3′ dinucleotideoverhangs was used. The sequence (5′ to 3′) of the guide/antisensestrand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 14228). The same base,sugar and phosphate modifications that were used for the active MSTNsiRNA duplex were used in the negative control siRNA. All siRNA singlestrands were fully assembled on solid phase using standardphospharamidite chemistry and purified over HPLC. Purified singlestrands were duplexed to get the double stranded siRNA. The passengerstrand contained two conjugation handles, a C6-NH₂ at the 5′ end and aC6-SH at the 3′ end. Both conjugation handles were connected to siRNApassenger strand via phosphodiester-inverted abasic-phosphodiesterlinker. Because the free thiol was not being used for conjugation, itwas end capped with N-ethylmaleimide.

Antibody siRNA Conjugate Synthesis Using Bis-Maleimide (BisMal) Linker

Step 1: Antibody Reduction with TCEP

Antibody was buffer exchanged with 25 mM borate buffer (pH 8) with 1 mMDTPA and made up to 10 mg/ml concentration. To this solution, 4equivalents of TCEP in the same borate buffer were added and incubatedfor 2 hours at 37° C. The resultant reaction mixture was combined with asolution of BisMal-siRNA (1.25 equivalents) in pH 6.0 10 mM acetatebuffer at RT and kept at 4° C. overnight. Analysis of the reactionmixture by analytical SAX column chromatography showed antibody siRNAconjugate along with unreacted antibody and siRNA. The reaction mixturewas treated with 10 EQ of N-ethylmaleimide (in DMSO at 10 mg/mL) to capany remaining free cysteine residues.

Step 2: Purification

The crude reaction mixture was purified by AKTA Pure FPLC using anionexchange chromatography (SAX) method-1. Fractions containing DAR1 andDAR2 antibody-siRNA conjugates were isolated, concentrated and bufferexchanged with pH 7.4 PBS.

Anion Exchange Chromatography Method (SAX)-1.

Column: Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5 mm ID×15 cm, 13 um

Solvent A: 20 mM TRIS buffer, pH 8.0; Solvent B: 20 mM TRIS, 1.5 M NaCl,pH 8.0; Flow Rate: 6.0 ml/min

Gradient:

a. % A % B Column Volume b. 100 0 1 c. 81 19 0.5 d. 50 50 13 e. 40 600.5 f. 0 100 0.5 g. 100 0 2

Anion Exchange Chromatography (SAX) Method-2

Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm

Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80% 10 mM TRISpH 8, 20% ethanol, 1.5 M NaCl; Flow Rate: 0.75 ml/min

Gradient:

a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d. 11.00 40 60 e. 14.00 40 60f. 15.00 20 80 g. 16.00 90 10 h. 20.00 90 10

Step-3: Analysis of the Purified Conjugate

The purity of the conjugate was assessed by analytical HPLC using anionexchange chromatography method-2 (Table 25).

TABLE 25 SAX retention % purity (by Conjugate time (min) peak area)TfR1-Atrogin-1 DAR1 9.2 95 TfR1-MuRF1 DAR1 9.3 92 mTfR1-SC (DAR1) 8.9 76

In Vivo Study Design

The conjugates were assessed for their ability to mediate mRNAdownregulation of MuRF1 and Atrogin-1 in muscle (gastroc) in thepresence and absence of sciatic nerve denervation, in an in vivoexperiment (C57BL6 mice). Mice were dosed via intravenous (iv) injectionwith PBS vehicle control and the indicated ASCs and doses, see FIG. 38.Seven days post conjugate delivery, for groups 2-4, 6-8, 10-12 and14-16, leg muscle atrophy was induced by sciatic nerve denervation.Denervation was not induced for the control groups 1, 5, 9, 13, and 17.

For the Denervation procedure at day 7, mice were anesthesed (5%isoflurane) and administer a subcutaneous dose of 0.1 mg/kgBuprenorphine. The right dorsal pelvic region was shaved from thesciatic notch to the knee. The area was disinfected with alternatingalcohol and povidone-iodine. The sciatic notch was identified bypalpation and an incision made from the sciatic notch towards the knee,approximately lcm. The bicep femoris muscle was split to expose thesciatic nerve and about a lcm fragment was removed by cauterizing bothends. The muscle and skin were then sutured to close the incision. Theoperative limb was then inspected daily to observe the condition of thesurgical wound and observe the animal for overall health.

For groups 4, 8, 12, and 16 changes in leg muscle area were determined:The leg-to-be-measured were shaved and a line was drawn using indelibleink to mark region of measurement. Mice were restrained in a conerestraint and the right leg was held by hand. Digital calipers were usedto take one measurement on the sagittal plane and another on the coronalplane. The procedure was repeated twice per week. For all groups at thetime points indicated, gastrocnemius (gastroc) and heart muscle tissueswere harvested, weighed and snap-frozen in liquid nitrogen. mRNAknockdown in target tissue was determined using a comparative qPCR assayas described in the methods section. Total RNA was extracted from thetissue, reverse transcribed and mRNA levels were quantified using TaqManqPCR, using the appropriately designed primers and probes. PPIB(housekeeping gene) was used as an internal RNA loading control, resultswere calculated by the comparative Ct method, where the differencebetween the target gene Ct value and the PPIB Ct value (ΔCt) iscalculated and then further normalized relative to the PBS control groupby taking a second difference (ΔΔCt).

Quantitation of tissue siRNA concentrations was determined using astem-loop qPCR assay as described in the methods section. The antisensestrand of the siRNA was reverse transcribed using a TaqMan MicroRNAreverse transcription kit using a sequence-specific stem-loop RT primer.The cDNA from the RT step was then utilized for real-time PCR and Ctvalues were transformed into plasma or tissue concentrations using thelinear equations derived from the standard curves.

FIG. 39A shows a single treatment of 4.5 mg/kg (siRNA) of eitherAtrogin-1 siRNA or MuRF1 siRNA or a single dose of both siRNAs combinedresulted in up to 75% downregulation of each target in thegastrocnemius.

FIG. 39B shows mRNA knockdown of both targets in gastrocnemius ismaintained at 75% in the intact leg out to 37 days post ASC dose.

In the denerved leg, Atrogin1 mRNA knockdown is maintained 3 days postdenervation, but is reduced to 20% by 10 days post denervation and to 0%by 30 days post denervation. MuRF1 mRNA knockdown in the denerved leg isenhanced to 80-85% 3 days post denervation, but is reduced to 50% by 10days post denervation and to 40% by 30 days post denervation (FIG. 39C).

The mRNA knockdown of each target was not impacted by the knockdown ofthe other target when treated with the combination of both siRNAs (FIG.39D).

Based on leg muscle area measurements, siRNA-mediated downregulation ofMuRF1 and the combination of MuRF1 and Atrogin-1 reduceddenervation-induced muscle wasting by up to 30%. Treatment with MuRF1siRNA alone showed similar responses than treatment with the combinationof MuRF1 and Atrogin-1. Downregulation of Atrogin-1 alone had nosignificant effect on leg muscle area. The statistical analysis comparedthe treatment groups to the scramble siRNA control group using a Welch'sTTest. See FIG. 39E.

Based on the Gastrocnemius weight only MuRF1 showed statisticallysignificant differences from the scramble siRNA control group. Similarto the results obtained by measuring leg muscle area, downregulation ofMuRF1 showed an up to 35% reduction in denervation-induced musclewasting. These results agree with effects of MuRF1 knock out in mice(Bodine et al., Science 291, 2001). See FIG. 39F.

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing thedisclosure. It is intended that the following claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

What is claimed is:
 1. A method of treating muscle atrophy in a subjectin need thereof comprising administering to said subject apolynucleotide conjugate comprising an anti-transferrin receptorantibody or antigen-binding fragment thereof conjugated to apolynucleotide that mediates RNA interference against the Atrogin-1 mRNApreferentially in a muscle cell, thereby treating muscle atrophy in saidsubject.
 2. The method of claim 1, wherein the polynucleotide is asingle stranded antisense polynucleotide or a double strandedpolynucleotide.
 3. The method of claim 1, wherein the polynucleotidecomprises at least one 2′ modified nucleotide, at least one modifiedinternucleotide linkage, or at least one inverted abasic moiety.
 4. Themethod of claim 1, wherein the mediation of RNA interference against theAtrogin-1 mRNA modulates muscle atrophy or myotonic dystrophy in saidsubject.
 5. The method of claim 1, wherein the anti-transferrin receptorantibody or antigen-binding fragment thereof binds to a transferrinreceptor on the cell surface of the muscle cell.
 6. The method of claim1, wherein the muscle cell is a skeletal muscle cell or a cardiac musclecell.
 7. The method of claim 1, wherein the polynucleotide hybridizes toa target sequence of the Atrogin-1 mRNA and mediates RNA interferenceagainst the Atrogin-1 mRNA via RNase H activity in the muscle cell. 8.The method of claim 1, wherein the polynucleotide hybridizes to at least8 contiguous bases of the target sequence of the Atrogin-1 mRNA.
 9. Themethod of claim 1, wherein the polynucleotide is from about 8 to about50 nucleotides in length or from about 10 to about 30 nucleotides inlength.
 10. The method of claim 1, wherein the oligonucleotide conjugatecomprises a linker connecting the anti-transferrin receptor antibody orantigen-binding fragment thereof to the polynucleotide.
 11. The methodof claim 3, wherein the at least one 2′ modified nucleotide: comprises2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,2′-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified nucleotide; comprises locked nucleic acid (LNA) orethylene nucleic acid (ENA); or comprises a combination thereof.
 12. Themethod of claim 3, wherein the at least one modified internucleotidelinkage comprises a phosphorothioate linkage or a phosphorodithioatelinkage.
 13. The method of claim 3, wherein the polynucleotide comprises3 or more 2′ modified nucleotides selected from 2′-O-methyl and2′-deoxy-2′-fluoro.
 14. The method of claim 1, wherein thepolynucleotide conjugate has a drug/polynucleotide to antibody ratio offrom about 1 to about
 4. 15. The method of claim 1, wherein thepolynucleotide comprises a 5′-terminal vinylphosphonate modifiednucleotide.
 16. The method of claim 1, wherein the anti-transferrinreceptor antibody or antigen-binding fragment thereof comprises ahumanized antibody or antigen-binding fragment thereof, chimericantibody or antigen-binding fragment thereof, monoclonal antibody orantigen-binding fragment thereof, monovalent Fab′, divalent Fab2,single-chain variable fragment (scFv), diabody, minibody, nanobody,single-domain antibody (sdAb), or camelid antibody or antigen-bindingfragment thereof.
 17. The method of claim 4, wherein the muscle atrophyis associated with myotonic dystrophy.
 18. The method of claim 4,wherein the muscle atrophy is caused by disuse, starvation, cancer,diabetes, renal failure, or treatment with glucocorticoids.
 19. Themethod of claim 1, wherein the polynucleotide conjugate is administeredparenterally, orally, intranasally, buccally, rectally, ortransdermally.
 20. A method of modulating muscle atrophy in a subject inneed thereof comprising administering to said subject a polynucleotideconjugate comprising an anti-transferrin receptor antibody orantigen-binding fragment thereof conjugated to a polynucleotide thatmediates RNA interference against the Atrogin-1 mRNA preferentially in amuscle cell in said subject, thereby modulating muscle atrophy in saidsubject.
 21. A method of modulating Atrogin-1 mRNA in a muscle cell of asubject comprising administering to said subject a polynucleotideconjugate comprising an anti-transferrin receptor antibody orantigen-binding fragment thereof conjugated to a polynucleotide thatmediates RNA interference against the Atrogin-1 mRNA in the muscle cell,thereby modulating Atrogin-1 mRNA in the muscle cell of said subject.22. A method of alleviating muscle atrophy in a subject suffering from amuscle disorder comprising administering to said subject apolynucleotide conjugate comprising an anti-transferrin receptorantibody or antigen-binding fragment thereof conjugated to apolynucleotide that mediates RNA interference against the Atrogin-1 mRNApreferentially in a muscle cell in said subject, thereby alleviatingmuscle atrophy in said subject.